Rat - MCAo 30 min Rat - MCAo 60 min Mouse - distal MCAo Mouse - proximal MCAo Marmoset - transient MCAo Marmoset - permanent MCAo

**CONTROLATERAL IPSILATERAL**

week after surgery.

Week 1 Week 2 Week 3 Week 4 Week 9

Week 1 Week 2 Week 3 Week 4 Week 9

**CONTROLATERAL**

on the ipsilateral side of the lesion and might be due, at least partly, to dizziness of the animal due to surgery and/or the anesthesia. With respect to the time to remove the adhesive, a bilateral motor coordination deficit is observed whatever the duration of the occlusion. Of note, it has been demonstrated in this same model of cerebral ischemia a spontaneous functional recovery on the ipsilateral side, while controlateral time to remove the adhesive remains hardly affected up to 4 weeks after surgery (Bihel et al.).

### **3.2 Correlations between brain histological damage and deficit in the adhesive removal test**

Correlations between the cortical and striatal histological lesions and the ischemia-induced behavioral impairments in the adhesive removal test have been well investigated in the literature, mostly in rodents (Grabowski et al., 1991; Hudzik et al., 2000; Hunter et al., 1998; Rogers et al., 1997; Virley et al., 2000). We and other authors have demonstrated a close correlation between contralateral contact and removal latencies on this task and abnormal changes in the ipsilateral caudate putamen, lower parietal cortex and forelimb cortex following transient MCAo (30; 60 or 90 min; see Figure 5). Each of these regions of interest contributed to functional impairments on this task across an extended time course (up to several months post-ischemia) (Virley et al., 2000). As regards to the final cortical damage, it seems particularly correlated to both transient and long-lasting sensorimotor deficits measured by adhesive removal test. Conversely, the final striatal lesion appears to be consistently related to the adhesive removal motor deficits (time to remove) (Freret et al., 2006).

Fig. 5. Schemes showing extent of the lesion after distal and proximal models of Middle Cerebral Artery occlusion (MCAo) in rodents and marmoset. Distal MCAo refers to the occlusion of the artery in its distal portion, *i.e.* after lenticulostriate branches (usually

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 59

al., 2008). This test has also proven to be useful for cellular therapies (Minnerup et al., 2011; Shen et al., 2007), or other pharmacological approaches (testosterone injection – (Morris et al., 2010)), Thymosin beta4 I – (Pan et al., 2005)). In mice, Rehni et al., 2007 (Rehni et al., 2007) showed that intracerebroventricular administration of stem cells derived from amniotic fluid is beneficial for adhesive removal after 60-min MCAo in mice. In marmosets, till now only few studies have assessed the effects of pharmacological agents on adhesive

Overall, those results argue in favor of the use of the adhesive removal test to accurately assess the effects of pharmacological agents on functional outcome. Indeed, this task is suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote

Data reported here show that adhesive removal test *i.* can be easily performed in most species used in experimental research *ii.* is powerful to point out functional deficits on a long-term duration after brain injury. This is a particularly interesting point because it allows assessing efficiency of new therapeutic strategies. Those deficits are in human patients those for which no real efficient therapy exists, excepted rehabilitative strategies like kinesiotherapy. Measuring their importance in animals give models to go further in

Contact and removal times give complementary information about the nervous system deficiency. Although the measurement of contact time is not a perfect reflect of sensory system functioning, because for obvious reasons it is related to a motor action (shaking paw, bringing paw to the mouth), it reflects nevertheless a sensory system stimulation. The animal feels or not that something is stuck on its paw and reacts by a movement. From experimental data obtained with adhesive removal, it is not possible to distinguish if the deficit in contact time is related to a primary somatosensory dysfunction (Ward et al., 1997), a sensory hemi-neglect (related to attentional deficit of a body part and involving striatum (Heilman et al., 2000; Reep et al., 2004)), or a tactile extinction phenomenon (related to

Removal of the tape is a rather tricky task for all the animals presented here because it is not *per se* a natural situation. Rodents and marmosets use their mouth to do it, and amazingly after stroke rats often hold their deficient paw with the intact one to bring it to the mouth as if they were not able to maintain at the right position the deficient/controlateral hand. The data presented here show that for equivalent brain lesions (parietal cortex and striatum affected by intraluminal model of stroke) all species tested display long-lasting deficits in removal time, at least at the dates tested, which is however rather long compared to delays usually assessed in stroke investigations. Time to contact the contralateral adhesive is also increased for few weeks after stroke, and comes back to normal levels for the less severe brain injury, while it stays abnormal for more severe injuries. It is important to note that the severity of the deficit is quite well related with the extent of the lesion, but also to the structures affected. When the parietal cortex alone is affected (30-min MCAo in rat, distal model in mouse), the deficit is less severe than when other structures are also involved in the lesioned area. Indeed, when the striatum is also affected (60-min MCAo in rat, proximal

removal test after stroke, but much on Parkinson's disease (Annett et al., 1994).

**4. General overview and limitations of the adhesive removal test**

regeneration.

researches on brain injury recovery.

bilateral stimulation (Schallert &Whishaw, 1984)).

obtained by electrocoagulation or administration of pharmacological agent, *e.g.* endothelin-1 (Virley et al., 2004)), while proximal MCAo refers to an occlusion at the origin of the artery (intraluminal approach). Extent of the lesion has been drawn from our data and literature. Of note, these schemes are not fully representative of all experimental models of middle cerebral artery ischemia, but give an overview of most frequently obtained brain lesion.

On a side point, the potential relationship between impairments in the adhesive removal test and brain regions that undergo secondary degeneration has been less investigated so far.  These regions that do not belong to the territory of the MCA, are connected to the primary lesion site. As regards to secondary thalamus shrinkage, retrograde as well as anterograde degeneration due, respectively, to cortical lesion (Iizuka et al., 1990) (Wallerian) and to basal ganglia lesion and/or extensive vasogenic edema (Dihne et al., 2002) are thought to be responsible. Whether the extent of these degenerative changes is or not directly correlated to the size of primary lesion is still under controversy (Freret et al., 2006; Iizuka et al., 1990). No matter how, in a rat stroke model, the final thalamic atrophy appears to be correlated with ischemia-induced deficits observed in the adhesive-removal test (especially its sensory component) (Freret et al., 2006). This correlation suggests that the thalamus makes, albeit moderately, a significant contribution to the ischemia-induced longlasting somatosensory (ipsilateral bias) and sensorimotor deficits (dexterity alteration). These findings are in agreement with the known involvement of thalamic ventroposterior nuclei in the somatosensory pathways and sensory processing (Tracey &Waite, 1995). In addition, correlations appeared at the late chronic stage (*i.e.*, several weeks after ischemic insult, which is consistent with the delay of thalamic shrinkage development; (Fujie et al., 1990; Jones &Schallert, 1992). Regarding substantia nigra, damage to this brain structure is also correlated with contralateral deficits in the adhesive removal test (Virley et al., 2000). Finally, talking about correlations between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, one must keep in mind that although the presence of such a correlation would make the interpretation of performances in this behavioral test easier, the absence of a linear relationship between both parameters does not mean the absence of involvement of the structure in the behavioral task. Indeed, one can imagine that beyond a single and direct relationship between one or several brain structures and the behavioral performances, those latter might depend upon the integrity of a cerebral network that can be influenced by others brain structures. If so, a lesion limited only to this cerebral network or to the related brain structures will have consequences on behavioral performances; even though the one or the other remains unaffected.

### **3.3 Sensitivity of the adhesive removal test to pharmacological treatment**

In line with this consideration of a relationship between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, our group have demonstrated that a delayed and chronic administration of deferoxamine (an iron chelator) reduces the secondary thalamic atrophy and improves functional recovery in the adhesive removal test after focal ischemia in the rat (Freret et al., 2006). Similarly, a neuroprotective effect of D-JNKi (a peptide inhibitor of JNK (c-Jun-N-terminal kinase)) have been highlighted and corroborated with adhesive removal improvement in the rat (Esneault et

obtained by electrocoagulation or administration of pharmacological agent, *e.g.* endothelin-1 (Virley et al., 2004)), while proximal MCAo refers to an occlusion at the origin of the artery (intraluminal approach). Extent of the lesion has been drawn from our data and literature. Of note, these schemes are not fully representative of all experimental models of middle cerebral artery ischemia, but give an overview of most frequently obtained brain lesion.

On a side point, the potential relationship between impairments in the adhesive removal test and brain regions that undergo secondary degeneration has been less investigated so far.  These regions that do not belong to the territory of the MCA, are connected to the primary lesion site. As regards to secondary thalamus shrinkage, retrograde as well as anterograde degeneration due, respectively, to cortical lesion (Iizuka et al., 1990) (Wallerian) and to basal ganglia lesion and/or extensive vasogenic edema (Dihne et al., 2002) are thought to be responsible. Whether the extent of these degenerative changes is or not directly correlated to the size of primary lesion is still under controversy (Freret et al., 2006; Iizuka et al., 1990). No matter how, in a rat stroke model, the final thalamic atrophy appears to be correlated with ischemia-induced deficits observed in the adhesive-removal test (especially its sensory component) (Freret et al., 2006). This correlation suggests that the thalamus makes, albeit moderately, a significant contribution to the ischemia-induced longlasting somatosensory (ipsilateral bias) and sensorimotor deficits (dexterity alteration). These findings are in agreement with the known involvement of thalamic ventroposterior nuclei in the somatosensory pathways and sensory processing (Tracey &Waite, 1995). In addition, correlations appeared at the late chronic stage (*i.e.*, several weeks after ischemic insult, which is consistent with the delay of thalamic shrinkage development; (Fujie et al., 1990; Jones &Schallert, 1992). Regarding substantia nigra, damage to this brain structure is also correlated with contralateral deficits in the adhesive removal test (Virley et al., 2000). Finally, talking about correlations between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, one must keep in mind that although the presence of such a correlation would make the interpretation of performances in this behavioral test easier, the absence of a linear relationship between both parameters does not mean the absence of involvement of the structure in the behavioral task. Indeed, one can imagine that beyond a single and direct relationship between one or several brain structures and the behavioral performances, those latter might depend upon the integrity of a cerebral network that can be influenced by others brain structures. If so, a lesion limited only to this cerebral network or to the related brain structures will have consequences on

behavioral performances; even though the one or the other remains unaffected.

**3.3 Sensitivity of the adhesive removal test to pharmacological treatment**

In line with this consideration of a relationship between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, our group have demonstrated that a delayed and chronic administration of deferoxamine (an iron chelator) reduces the secondary thalamic atrophy and improves functional recovery in the adhesive removal test after focal ischemia in the rat (Freret et al., 2006). Similarly, a neuroprotective effect of D-JNKi (a peptide inhibitor of JNK (c-Jun-N-terminal kinase)) have been highlighted and corroborated with adhesive removal improvement in the rat (Esneault et al., 2008). This test has also proven to be useful for cellular therapies (Minnerup et al., 2011; Shen et al., 2007), or other pharmacological approaches (testosterone injection – (Morris et al., 2010)), Thymosin beta4 I – (Pan et al., 2005)). In mice, Rehni et al., 2007 (Rehni et al., 2007) showed that intracerebroventricular administration of stem cells derived from amniotic fluid is beneficial for adhesive removal after 60-min MCAo in mice. In marmosets, till now only few studies have assessed the effects of pharmacological agents on adhesive removal test after stroke, but much on Parkinson's disease (Annett et al., 1994).

Overall, those results argue in favor of the use of the adhesive removal test to accurately assess the effects of pharmacological agents on functional outcome. Indeed, this task is suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote regeneration.

### **4. General overview and limitations of the adhesive removal test**

Data reported here show that adhesive removal test *i.* can be easily performed in most species used in experimental research *ii.* is powerful to point out functional deficits on a long-term duration after brain injury. This is a particularly interesting point because it allows assessing efficiency of new therapeutic strategies. Those deficits are in human patients those for which no real efficient therapy exists, excepted rehabilitative strategies like kinesiotherapy. Measuring their importance in animals give models to go further in researches on brain injury recovery.

Contact and removal times give complementary information about the nervous system deficiency. Although the measurement of contact time is not a perfect reflect of sensory system functioning, because for obvious reasons it is related to a motor action (shaking paw, bringing paw to the mouth), it reflects nevertheless a sensory system stimulation. The animal feels or not that something is stuck on its paw and reacts by a movement. From experimental data obtained with adhesive removal, it is not possible to distinguish if the deficit in contact time is related to a primary somatosensory dysfunction (Ward et al., 1997), a sensory hemi-neglect (related to attentional deficit of a body part and involving striatum (Heilman et al., 2000; Reep et al., 2004)), or a tactile extinction phenomenon (related to bilateral stimulation (Schallert &Whishaw, 1984)).

Removal of the tape is a rather tricky task for all the animals presented here because it is not *per se* a natural situation. Rodents and marmosets use their mouth to do it, and amazingly after stroke rats often hold their deficient paw with the intact one to bring it to the mouth as if they were not able to maintain at the right position the deficient/controlateral hand.

The data presented here show that for equivalent brain lesions (parietal cortex and striatum affected by intraluminal model of stroke) all species tested display long-lasting deficits in removal time, at least at the dates tested, which is however rather long compared to delays usually assessed in stroke investigations. Time to contact the contralateral adhesive is also increased for few weeks after stroke, and comes back to normal levels for the less severe brain injury, while it stays abnormal for more severe injuries. It is important to note that the severity of the deficit is quite well related with the extent of the lesion, but also to the structures affected. When the parietal cortex alone is affected (30-min MCAo in rat, distal model in mouse), the deficit is less severe than when other structures are also involved in the lesioned area. Indeed, when the striatum is also affected (60-min MCAo in rat, proximal

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 61

study reporting the usefulness of the tongue protrusion test in a rat model of proximal cerebral ischemia (Gulyaeva et al., 2003). Furthermore, it suggests that permanent distal stroke could be a relevant model of oral and facial impairments, which are also tremendous

To conclude on the usefulness of the adhesive removal test, one must admit that this test:

3. is useful to assess recovery, since it is capable of measuring long-lasting deficits,

2. is an accurate test to assess somatosensory and motor dysfunctions, even if they are

4. allows for longitudinal studies through adaptation of the size of the adhesive tape according to the age of the individual tested. Indeed, as previously described, we adapted the adhesive removal test to in 20 days old rat pups by reducing the size of the

5. Finally, the adhesive removal test allows for interspecies comparison (marmosets, rats, dogs and mice), as strongly advised by the expert committees for preclinical studies.

Annett LE, Martel FL, Rogers DC, Ridley RM, Baker HF, Dunnett SB. Behavioral assessment

Annett LE, Rogers DC, Hernandez TD, Dunnett SB. Behavioural analysis of unilateral monoamine depletion in the marmoset. Brain 1992;115 ( Pt 3):825-56. Baumann CR, Kilic E, Petit B, Werth E, Hermann DM, Tafti M, Bassetti CL. Sleep EEG

Bihel E, Pro-Sistiaga P, Letourneur A, Toutain J, Saulnier R, Insausti R, Bernaudin M,

Bouet V, Boulouard M, Toutain J, Divoux D, Bernaudin M, Schumann-Bard P, Freret T. The

Bouet V, Freret T, Ankri S, Bezault M, Renolleau S, Boulouard M, Jacotot E, Chauvier D,

Bouet V, Freret T, Toutain J, Divoux D, Boulouard M, Schumann-Bard P. Sensorimotor and

Bulman-Fleming MB, Bryden MP, Rogers TT. Mouse paw preference: effects of variations in

Denes G, Semenza C, Stoppa E, Lis A. Unilateral spatial neglect and recovery from

ischemic stroke with reperfusion in the rat. Behav Brain Res 2010.

lesions of the nigrostriatal pathway. Exp Neurol 1994;125(2):228-46.

cortical lesions. Sleep 2006;29(10):1339-44.

Nat Protoc 2009;4(10):1560-4.

Neurol 2007;203(2):555-67.

investigations. J Cereb Blood Flow Metab;30(2):273-85.

testing protocol. Behav Brain Res 1997;86(1):79-87.

hemiplegia: a follow-up study. Brain 1982;105 (Pt 3):543-52.

of the effects of embryonic nigral grafts in marmosets with unilateral 6-OHDA

changes after middle cerebral artery infarcts in mice: different effects of striatal and

Roussel S, Touzani O. Permanent or transient chronic ischemic stroke in the nonhuman primate: behavioral, neuroimaging, histological, and immunohistochemical

adhesive removal test: a sensitive method to assess sensorimotor deficits in mice.

Schumann-Bard P. Predicting sensorimotor and memory deficits after neonatal

cognitive deficits after transient middle cerebral artery occlusion in the mouse. Exp

1. is relevant because it was developed in relation to clinical evaluations,

problems in human stroke.

**5. Conclusion**

very tiny,

adhesive tape.

**6. References**

model in mouse, both models in marmoset), the recovery is even longer and most of the time not complete within the delays tested.

Mechanisms implied in the recovery of the ability to remove the adhesive tape are not well determined but some of them have to be considered: structural modifications are undoubted (sprouting, synaptogenesis,..), functional modifications (synaptic plasticity, use of brain areas close to the lesion site to fulfill the roles of destroyed tissue…), sensory substitution (increasing role of deep cutaneous modality to replace for the superficial one…), or even behavioral strategies (as for instance, the rat holding the deficient paw with the intact paw to bring it to its mouth).

### **4.1 Sensory hemi-neglect or tactile extinction syndrome**

In rats, a 60-min MCAo induces a long-lasting motor coordination deficit on the contralateral side, reflecting a failure to respond to a novel tactile stimulus. Whether this phenomenon reflects either a primary somatosensory dysfunction (Ward et al., 1997), a truly sensory hemi-neglect (*i.e.*, an attention deficit, as it is classically invoked in the clinic; (Heilman et al., 2000)), or a tactile extinction syndrome (*i.e.*, an interhemispheric perceptual interaction between both stimuli; (Schallert &Whishaw, 1984) is hard to say. Indeed, such duration of occlusion induces a lesion that includes the primary sensory cortical area (S1FL) and, in a secondary degenerative manner, part of the thalamus. These two regions are involved in the somatosensory information processing. On the other hand, it has been shown that unilateral lesions of the posterior parietal cortex and of the dorsocentral striatum (i.e., two structures that are at least partly affected by ischemia) result in multimodal neglect in the rat (Reep et al., 2004). Clinical studies on healthy subjects have reported that the vigilance component of attention to sensory stimuli involves, at least in part, the parietal cortex (Pardo et al., 1991). Thus, a lesion of the parietal cortex could also contribute to a sensory neglect syndrome in the rat after ischemia. An alteration of the cortical– basalganglia–thalamic network could lead to a sensory neglect syndrome in the rat. Nevertheless, a tactile extinction syndrome occurs frequently in stroke patients with right cerebral hemisphere damage (Rose et al., 1994), and the presence of neglect is not only debilitating for patient's ability for independent daily life but is also a significant predictor of poor outcome for recovery from hemiplegia in stroke patients (Denes et al., 1982). The longlasting correlation described between the contralateral time to contact and the final cortical damage (Freret et al., 2006) reinforces the idea that this structure makes significant contribution to the sensory impairment observed after ischemia (Virley et al., 2000).

#### **4.2 Facial and/or limb impairments**

After d-MCAo in mice, even though the extension of the lesion is relatively limited, the adhesive removal test is efficient to highlight sensorimotor contralateral impairment (time to removal) 3 weeks after the surgery. This deficit may combine both sensory feedback alterations and motor coordination that are not attributable to postural bias (Schallert et al., 1983). Given the spatial distribution of the cerebral lesion (Figure 5), this impairment might also reflect in this case a face-related somatososensory perception alteration (*i.e.*, a difficulty for the animal to sense the adhesive with its ipsilateral whiskers when it approaches the tape to his face and/or with its tongue when he licks the adhesive in order to facilitate the removal) rather than a forelimb motor or sensory alteration (because the related brain region, *i.e.*, S1 FL, is mainly spared by ischemia). This hypothesis is in accordance with a study reporting the usefulness of the tongue protrusion test in a rat model of proximal cerebral ischemia (Gulyaeva et al., 2003). Furthermore, it suggests that permanent distal stroke could be a relevant model of oral and facial impairments, which are also tremendous problems in human stroke.

### **5. Conclusion**

60 Advances in the Preclinical Study of Ischemic Stroke

model in mouse, both models in marmoset), the recovery is even longer and most of the

Mechanisms implied in the recovery of the ability to remove the adhesive tape are not well determined but some of them have to be considered: structural modifications are undoubted (sprouting, synaptogenesis,..), functional modifications (synaptic plasticity, use of brain areas close to the lesion site to fulfill the roles of destroyed tissue…), sensory substitution (increasing role of deep cutaneous modality to replace for the superficial one…), or even behavioral strategies (as for instance, the rat holding the deficient paw with the intact paw

In rats, a 60-min MCAo induces a long-lasting motor coordination deficit on the contralateral side, reflecting a failure to respond to a novel tactile stimulus. Whether this phenomenon reflects either a primary somatosensory dysfunction (Ward et al., 1997), a truly sensory hemi-neglect (*i.e.*, an attention deficit, as it is classically invoked in the clinic; (Heilman et al., 2000)), or a tactile extinction syndrome (*i.e.*, an interhemispheric perceptual interaction between both stimuli; (Schallert &Whishaw, 1984) is hard to say. Indeed, such duration of occlusion induces a lesion that includes the primary sensory cortical area (S1FL) and, in a secondary degenerative manner, part of the thalamus. These two regions are involved in the somatosensory information processing. On the other hand, it has been shown that unilateral lesions of the posterior parietal cortex and of the dorsocentral striatum (i.e., two structures that are at least partly affected by ischemia) result in multimodal neglect in the rat (Reep et al., 2004). Clinical studies on healthy subjects have reported that the vigilance component of attention to sensory stimuli involves, at least in part, the parietal cortex (Pardo et al., 1991). Thus, a lesion of the parietal cortex could also contribute to a sensory neglect syndrome in the rat after ischemia. An alteration of the cortical– basalganglia–thalamic network could lead to a sensory neglect syndrome in the rat. Nevertheless, a tactile extinction syndrome occurs frequently in stroke patients with right cerebral hemisphere damage (Rose et al., 1994), and the presence of neglect is not only debilitating for patient's ability for independent daily life but is also a significant predictor of poor outcome for recovery from hemiplegia in stroke patients (Denes et al., 1982). The longlasting correlation described between the contralateral time to contact and the final cortical damage (Freret et al., 2006) reinforces the idea that this structure makes significant

contribution to the sensory impairment observed after ischemia (Virley et al., 2000).

After d-MCAo in mice, even though the extension of the lesion is relatively limited, the adhesive removal test is efficient to highlight sensorimotor contralateral impairment (time to removal) 3 weeks after the surgery. This deficit may combine both sensory feedback alterations and motor coordination that are not attributable to postural bias (Schallert et al., 1983). Given the spatial distribution of the cerebral lesion (Figure 5), this impairment might also reflect in this case a face-related somatososensory perception alteration (*i.e.*, a difficulty for the animal to sense the adhesive with its ipsilateral whiskers when it approaches the tape to his face and/or with its tongue when he licks the adhesive in order to facilitate the removal) rather than a forelimb motor or sensory alteration (because the related brain region, *i.e.*, S1 FL, is mainly spared by ischemia). This hypothesis is in accordance with a

time not complete within the delays tested.

**4.1 Sensory hemi-neglect or tactile extinction syndrome**

to bring it to its mouth).

**4.2 Facial and/or limb impairments**

To conclude on the usefulness of the adhesive removal test, one must admit that this test:

### **6. References**

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 63

Iizuka H, Sakatani K, Young W. Neural damage in the rat thalamus after cortical infarcts.

Ishibashi S, Kuroiwa T, Endo S, Okeda R, Mizusawa H. Neurological dysfunctions versus

Jones TA, Schallert T. Subcortical deterioration after cortical damage: effects of diazepam and relation to recovery of function. Behav Brain Res 1992;51(1):1-13. Komotar RJ, Kim GH, Sughrue ME, Otten ML, Rynkowski MA, Kellner CP, Hahn DK,

Marshall JW, Ridley RM. Assessment of functional impairment following permanent middle

Minnerup J, Kim JB, Schmidt A, Diederich K, Bauer H, Schilling M, Strecker JK, Ringelstein

Modo M, Stroemer RP, Tang E, Veizovic T, Sowniski P, Hodges H. Neurological sequelae

Morris DC, Chopp M, Zhang L, Lu M, Zhang ZG. Thymosin beta4 improves functional

Pan Y, Zhang H, Acharya AB, Patrick PH, Oliver D, Morley JE. Effect of testosterone on

Pardo JV, Fox PT, Raichle ME. Localization of a human system for sustained attention by

Quaranta A, Siniscalchi M, Frate A, Vallortigara G. Paw preference in dogs: relations between lateralised behaviour and immunity. Behav Brain Res 2004;153(2):521-5. Reep RL, Corwin JV, Cheatwood JL, Van Vleet TM, Heilman KM, Watson RT. A rodent

Rehni AK, Singh N, Jaggi AS, Singh M. Amniotic fluid derived stem cells ameliorate focal

Rogers DC, Campbell CA, Stretton JL, Mackay KB. Correlation between motor impairment

Rose L, Bakal DA, Fung TS, Farn P, Weaver LE. Tactile extinction and functional status after

Schaar KL, Brenneman MM, Savitz SI. Functional assessments in the rodent stroke model.

Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST. CNS plasticity and assessment

stroke. A preliminary investigation. Stroke 1994;25(10):1973-6.

positron emission tomography. Nature 1991;349(6304):61-4.

regional infarction volume after focal ischemia in Mongolian gerbils. Stroke

Merkow MB, Garrett MC, Starke RM, Connolly ES. Neurologic assessment of somatosensory dysfunction following an experimental rodent model of cerebral

cerebral artery occlusion in a non-human primate species. Neurodegeneration

EB, Sommer C, Scholer HR, Schabitz WR. Effects of neural progenitor cells on sensorimotor recovery and endogenous repair mechanisms after photothrombotic

and long-term behavioural assessment of rats with transient middle cerebral artery

neurological outcome in a rat model of embolic stroke. Neuroscience

functional recovery in a castrate male rat stroke model. Brain Res 2005;1043(1-

model for investigating the neurobiology of contralateral neglect. Cogn Behav

cerebral ischaemia-reperfusion injury induced behavioural deficits in mice. Behav

and infarct volume after permanent and transient middle cerebral artery occlusion

of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical

Stroke 1990;21(5):790-4.

ischemia. Nat Protoc 2007;2(10):2345-7.

stroke. Stroke 2011;42(6):1757-63.

occlusion. J Neurosci Methods 2000;104(1):99-109.

in the rat. Stroke 1997;28(10):2060-5; discussion 6.

2003;34(6):1501-6.

1996;5(3):275-86.

2010;169(2):674-82.

Neurol 2004;17(4):191-4.

Brain Res 2007;183(1):95-100.

Exp Transl Stroke Med 2010;2(1):13.

2):195-204.

Dihne M, Grommes C, Lutzenburg M, Witte OW, Block F. Different mechanisms of

Esneault E, Castagne V, Moser P, Bonny C, Bernaudin M. D-JNKi, a peptide inhibitor of c-

Fleming SM, Salcedo J, Fernagut PO, Rockenstein E, Masliah E, Levine MS, Chesselet MF.

Freret T, Bouet V, Leconte C, Roussel S, Chazalviel L, Divoux D, Schumann-Bard P,

Freret T, Chazalviel L, Roussel S, Bernaudin M, Schumann-Bard P, Boulouard M. Long-term

Freret T, Valable S, Chazalviel L, Saulnier R, Mackenzie ET, Petit E, Bernaudin M,

Fujie W, Kirino T, Tomukai N, Iwasawa T, Tamura A. Progressive shrinkage of the thalamus following middle cerebral artery occlusion in rats. Stroke 1990;21(10):1485-8. Grabowski M, Nordborg C, Johansson BB. Sensorimotor performance and rotation correlate

Guegan C, Braudeau J, Couriaud C, Dietz GP, Lacombe P, Bahr M, Nosten-Bertrand M,

Hudzik TJ, Borrelli A, Bialobok P, Widzowski D, Sydserff S, Howell A, Gendron P, Corbett

Iadecola C, Zhang F, Casey R, Nagayama M, Ross ME. Delayed reduction of ischemic brain

artery occlusion in the rat. Pharmacol Biochem Behav 2000;65(3):553-62. Hunter AJ, Mackay KB, Rogers DC. To what extent have functional studies of ischaemia in

transcriptional regulatory mechanisms. Neurobiol Dis 2006;22(1):177-86. Gulyaeva N, Thompson C, Shinohara N, Lazareva N, Onufriev M, Stepanichev M, Moiseeva

Usefulness of adhesive removal test. Behav Neurosci 2009;123(1):224-30. Freret T, Bouet V, Toutain J, Saulnier R, Pro-Sistiaga P, Bihel E, Mackenzie ET, Roussel S,

Stroke 2002;33(12):3006-11.

2006:in press.

2000;20(4):463-70.

Pharmacol Sci 1998;19(2):59-66.

gene. J Neurosci 1997;17(23):9157-64.

ischemia in rats. Neuroscience 2008;152(2):308-20.

human alpha-synuclein. J Neurosci 2004;24(42):9434-40.

human primate. J Cereb Blood Flow Metab 2008;28(4):786-96.

ischemia in the rat. Eur J Neurosci 2006;23(7):1757-65.

hypertensive rat. Brain Res 1991;547(2):249-57.

secondary neuronal damage in thalamic nuclei after focal cerebral ischemia in rats.

Jun N-terminal kinase, promotes functional recovery after transient focal cerebral

Early and progressive sensorimotor anomalies in mice overexpressing wild-type

Boulouard M. Behavioral deficits after distal focal cerebral ischemia in mice:

Schumann-Bard P, Touzani O. Intraluminal thread model of focal stroke in the non-

functional outcome following transient middle cerebral artery occlusion in the rat - Correlation between brain damage and behavioral impairment. Behav Neurosci

Boulouard M, Schumann-Bard P. Delayed administration of deferoxamine reduces brain damage and promotes functional recovery after transient focal cerebral

to lesion size in right but not left hemisphere brain infarcts in the spontaneously

Onteniente B. PTD-XIAP protects against cerebral ischemia by anti-apoptotic and

Y, Fliss H, Hakim AM. Tongue protrusion: a simple test for neurological recovery in rats following focal cerebral ischemia. J Neurosci Methods 2003;125(1-2):183-93. Heilman KM, Valenstein E, Watson RT. Neglect and related disorders. Semin Neurol

D, Miller J, Palmer GC. Long-term functional end points following middle cerebral

animals been useful in the assessment of potential neuroprotective agents? Trends

injury and neurological deficits in mice lacking the inducible nitric oxide synthase

**4**

**Variations in Origin of Arteries Supplying**

Animal testing, also known as animal experimentation, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million used annually (Cohn, 2010). The number of mice and rats used in the United States alone in 2001 was 80 million. Most animals are euthanized after being used in an experiment

It includes pure research such as genetics, developmental biology, behavioral studies, as well as applied research such as biomedical research, xenotransplantation, drug testing and toxicology tests, including cosmetics testing. Animals are also used for education, breeding and defense research. The practice is regulated to various degrees in different

The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE. Aristotle (384–322 BCE) and Erasistratus (304–258 BCE) were among

The ability of humans to change the genetics of animals took a large step forwards in 1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice (Jaenisch & Mintz, 1974). This genetic research progressed rapidly and in 1996 Dolly the sheep was born, the first mammal to be cloned

Toxicology testing became important in the 20th century. In the 19th century laws regulating drugs were more relaxed. For example, in the U.S. the government could only ban a drug after a company had been prosecuted for selling products that harmed customers. In the 1960s, in reaction to the Thalidomide tragedy, further laws were passed requiring safety testing on pregnant animals before a drug can be sold (Burkholz, 1997). Albino rabbits are used in eye irritancy tests because rabbits have less tear flow than other animals and the lack of eye pigment in albinos make the effects easier to visualize. Rabbits

the first to perform experiments on living animals (Cohen & Loew, 1984).

are also frequently used for the production of polyclonal antibodies.

**1. Introduction**

(Carbone, 2004).

countries.

**1.1 Laboratory animals**

from an adult cell (Wilmut et al., 1997).

**the Brain in Rabbit and Their Impact**

**on Total Cerebral Ischemia**

Eva Petrovova and Lenka Luptakova

*The Slovak Republic*

David Mazensky, Jan Danko, Emil Pilipcinec,

*University of Veterinary Medicine and Pharmacy, Kosice*

ablation, parkinsonism and spinal cord injury. Neuropharmacology 2000;39(5):777- 87.

## **Variations in Origin of Arteries Supplying the Brain in Rabbit and Their Impact on Total Cerebral Ischemia**

David Mazensky, Jan Danko, Emil Pilipcinec, Eva Petrovova and Lenka Luptakova *University of Veterinary Medicine and Pharmacy, Kosice The Slovak Republic*

### **1. Introduction**

64 Advances in the Preclinical Study of Ischemic Stroke

Schallert T, Upchurch M, Lobaugh N, Farrar SB, Spirduso WW, Gilliam P, Vaughn D,

Schallert T, Upchurch M, Wilcox RE, Vaughn DM. Posture-independent sensorimotor

Schallert T, Whishaw IQ. Bilateral cutaneous stimulation of the somatosensory system in

Shen LH, Li Y, Chen J, Cui Y, Zhang C, Kapke A, Lu M, Savant-Bhonsale S, Chopp M. One-

STAIR. Recommendations for standards regarding preclinical neuroprotective and

STAIRIII. Recommendations for clinical trial evaluation of acute stroke therapies. Stroke

Starkey ML, Barritt AW, Yip PK, Davies M, Hamers FP, McMahon SB, Bradbury EJ.

Tamura A, Kirino T, Sano K, Takagi K, Oka H. Atrophy of the ipsilateral substantia nigra following middle cerebral artery occlusion in the rat. Brain Res 1990;510(1):154-7. Tracey DJ, Waite ME. Somatosensory System. In: Press A, editor. The rat nervous system

van Lookeren Campagne M, Thibodeaux H, van Bruggen N, Cairns B, Gerlai R, Palmer JT,

Virley D, Beech JS, Smart SC, Williams SC, Hodges H, Hunter AJ. A temporal MRI

hemidecorticate rats. Behav Neurosci 1984;98(3):518-40.

restorative drug development. Stroke 1999;30(12):2752-8.

2nd Edition. Sydney: Academic Press; 1995. p 689-700.

corticospinal tract in adult mice. Exp Neurol 2005;195(2):524-39.

cerebral ischemia. Proc Natl Acad Sci U S A 1999;96(22):12870-5.

a rodent model of focal ischemia. Stroke 2011;42(5):1437-44.

87.

Behav 1982;16(3):455-62.

2001;32(7):1598-606.

Biochem Behav 1983;18(5):753-9.

rats with stroke. Stroke 2007;38(7):2150-6.

ablation, parkinsonism and spinal cord injury. Neuropharmacology 2000;39(5):777-

Wilcox RE. Tactile extinction: distinguishing between sensorimotor and motor asymmetries in rats with unilateral nigrostriatal damage. Pharmacol Biochem

analysis of inter-hemispheric receptor asymmetries in neostriatum. Pharmacol

year follow-up after bone marrow stromal cell treatment in middle-aged female

Assessing behavioural function following a pyramidotomy lesion of the

Williams SP, Lowe DG. Evidence for a protective role of metallothionein-1 in focal

assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior. J Cereb Blood Flow Metab 2000;20(3):563-82. Ward NM, Sharkey J, Brown VJ. Assessment of sensorimotor neglect after occlusion of the middle cerebral artery in the rat. Behav Neurosci 1997;111(5):1133-45. Zhang L, Li Y, Zhang C, Chopp M, Gosiewska A, Hong KW. Delayed administration of

human umbilical tissue-derived cells improved neurological functional recovery in

### **1.1 Laboratory animals**

Animal testing, also known as animal experimentation, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million used annually (Cohn, 2010). The number of mice and rats used in the United States alone in 2001 was 80 million. Most animals are euthanized after being used in an experiment (Carbone, 2004).

It includes pure research such as genetics, developmental biology, behavioral studies, as well as applied research such as biomedical research, xenotransplantation, drug testing and toxicology tests, including cosmetics testing. Animals are also used for education, breeding and defense research. The practice is regulated to various degrees in different countries.

The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE. Aristotle (384–322 BCE) and Erasistratus (304–258 BCE) were among the first to perform experiments on living animals (Cohen & Loew, 1984).

The ability of humans to change the genetics of animals took a large step forwards in 1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice (Jaenisch & Mintz, 1974). This genetic research progressed rapidly and in 1996 Dolly the sheep was born, the first mammal to be cloned from an adult cell (Wilmut et al., 1997).

Toxicology testing became important in the 20th century. In the 19th century laws regulating drugs were more relaxed. For example, in the U.S. the government could only ban a drug after a company had been prosecuted for selling products that harmed customers. In the 1960s, in reaction to the Thalidomide tragedy, further laws were passed requiring safety testing on pregnant animals before a drug can be sold (Burkholz, 1997).

Albino rabbits are used in eye irritancy tests because rabbits have less tear flow than other animals and the lack of eye pigment in albinos make the effects easier to visualize. Rabbits are also frequently used for the production of polyclonal antibodies.

Variations in Origin of Arteries Supplying

**1.3.3 Truncus brachiocephalicus**

Fig. 1. Basis cordis

**1.3.4 Arteria carotis communis**

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 67

aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases and the origin of the a. carotis communis sinistra in 4% of cases from the arcus aortae. White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra and the a.

The truncus brachiocephalicus runs behind the cartilage of the first rib and the v. cava cranialis dextra on the left side of the trachea. Near the midline it courses to the right and then partly dorsally. Immediatelly after its origin gives off the a. carotis communis sinistra. The truncus brachiocephalicus is very short and divides into the a. carotis communis dextra and the a. subclavia dextra (Nejedlý, 1965). By other author the a. subclavia dextra was

described as the first branch of the truncus brachiocephalicus (Popesko et al., 1990).

7. a. subclavia sinistra; 8. truncus brachiocephalicus; 9. a. subclavia dextra; 10. truncus bicaroticus; 13. n. vagus dexter; 14. n. recurrens dexter; 15. n. recurrens sinister; 20. v. cava cranialis sinistra; 21. v. cava cranialis dextra; 22. v. cava caudalis; 23. v. azygos dextra; 24. truncus pulmonalis; 25. a. pulmonalis sinistra; 26. a. pulmonalis dextra; 27. vv. pulmonales; 28. atrium sinistrum; 29. atrium dextrum; 30. ventriculus sinister; 31. ventriculus dexter; 32. ligamentum arteriosum; 33. lymphonodi thoracici aortici; 34. a. bronchalis. v. Vena. Vv.

In the neck region the a. carotis communis is covered by m. sternohyoideus (musculus), more cranially also by the m. sternothyroideus. It lies on the lateral surface of the trachea. The a. carotis communis lies dorsally to the esophagus, then laterally to the larynx. It reaches the maxillar angle laterally from the cranial end of the m. sternohyoideus and under the parotid gland. In this area it divides into the a. carotis interna and a. carotis externa (Nejedlý, 1965).

Venae. n. Nervus. a. Arteria. Dorsal view (Popesko et al., 1990)

subclavia dextra et sinistra as independent branches of the arcus aortae.

### **1.2 Cerebral ischemia**

It is the deficiency of blood and metabolic substrates in the brain due to insufficient arterial supply or venous drainage, causing disruption of cerebral functions and partially reversible or irreversible damage to neurons.

Ischemia leads to alterations in brain metabolism, reduction in metabolic rates and energy crisis (Vespa et al., 2005).

There are two types of ischemia: focal ischemia, which is confined to a specific region of the brain; and global ischemia, which encompasses wide areas of brain tissue.

The main symptoms involve impairments in vision, body movement, and speaking. The causes of brain ischemia vary from sickle cell anemia to congenital heart defects. Symptoms of brain ischemia can include unconsciousness, blindness, problems with coordination, and weakness in the body. Other effects that may result from brain ischemia are stroke, cardiorespiratory arrest and irreversible brain damage.

An interruption of blood flow to the brain for more 10 seconds causes unconsciousness and an interruption in flow for more than a few minutes generally results in irreversible brain damage. In 1974, Hossmann and Zimmerman demonstrated that ischemia induced in mammalian brains for up to an hour can be at least partially recovered. Accordingly, this discovery raised the possibility of intervening after brain ischemia before the damage becomes irreversible.

 This is commonly caused by cardiac arrest. If sufficient circulation is restored within a short period of time, symptoms may be transient. However, if a significant amount of time passes before restoration, brain damage may be permanent. While reperfusion may be essential to protecting as much brain tissue as possible, it may also lead to reperfusion injury. Reperfusion injury is classified as the damage that ensues after restoration of blood supply to ischemic tissue.

### **1.3 Anatomy of the arteries supplying the brain in the rabbit**

### **1.3.1 Aorta ascendens**

The aorta ascendens arises almost linearly and dorsally in the midline. It extends from the cranial margin of the second rib cartilage to the cranial margin of the first rib cartilage. At first runs inside the pericard, left to beginning of the pulmonary trunk, to the left and cranially to the dorsal part of the right auricle and to the left and cranially to the right ventricle. Then it runs inside the thymus dorsally to the left side of the v. cava cranialis dextra (vena) and continues to the arcus aortae (Nejedlý, 1965).

### **1.3.2 Arcus aortae**

The arcus aortae is running from the right to the left transversally and also a little caudally from the point of the second thoracic vertebra. Its dorsal wall is convex, the ventral wall is concave. It lies to the left from the v. cava cranialis dextra. The left part turns around the bronchus principalis sinister and ends on the left side of the third thoracic vertebra behind the v. cava cranialis sinistra. Very close to the midline of the body from its arise to the right the truncus brachiocephalicus and to the left the a. subclavia sinistra (arteria) (Popesko et al., 1990; Fig. 1). The truncus brachiocephalicus, the a. carotis communis sinistra and the a. subclavia dextra as branches of the arcus aortae were described by Nejedlý (1965). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases and the origin of the a. carotis communis sinistra in 4% of cases from the arcus aortae. White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra and the a. subclavia dextra et sinistra as independent branches of the arcus aortae.

### **1.3.3 Truncus brachiocephalicus**

66 Advances in the Preclinical Study of Ischemic Stroke

It is the deficiency of blood and metabolic substrates in the brain due to insufficient arterial supply or venous drainage, causing disruption of cerebral functions and partially reversible

Ischemia leads to alterations in brain metabolism, reduction in metabolic rates and energy

There are two types of ischemia: focal ischemia, which is confined to a specific region of the

The main symptoms involve impairments in vision, body movement, and speaking. The causes of brain ischemia vary from sickle cell anemia to congenital heart defects. Symptoms of brain ischemia can include unconsciousness, blindness, problems with coordination, and weakness in the body. Other effects that may result from brain ischemia are stroke,

An interruption of blood flow to the brain for more 10 seconds causes unconsciousness and an interruption in flow for more than a few minutes generally results in irreversible brain damage. In 1974, Hossmann and Zimmerman demonstrated that ischemia induced in mammalian brains for up to an hour can be at least partially recovered. Accordingly, this discovery raised the possibility of intervening after brain ischemia before the damage

Global brain ischemia occurs when blood flow to the brain is halted or drastically reduced. This is commonly caused by cardiac arrest. If sufficient circulation is restored within a short period of time, symptoms may be transient. However, if a significant amount of time passes before restoration, brain damage may be permanent. While reperfusion may be essential to protecting as much brain tissue as possible, it may also lead to reperfusion injury. Reperfusion injury is classified as the damage that ensues after restoration of blood supply

The aorta ascendens arises almost linearly and dorsally in the midline. It extends from the cranial margin of the second rib cartilage to the cranial margin of the first rib cartilage. At first runs inside the pericard, left to beginning of the pulmonary trunk, to the left and cranially to the dorsal part of the right auricle and to the left and cranially to the right ventricle. Then it runs inside the thymus dorsally to the left side of the v. cava cranialis

The arcus aortae is running from the right to the left transversally and also a little caudally from the point of the second thoracic vertebra. Its dorsal wall is convex, the ventral wall is concave. It lies to the left from the v. cava cranialis dextra. The left part turns around the bronchus principalis sinister and ends on the left side of the third thoracic vertebra behind the v. cava cranialis sinistra. Very close to the midline of the body from its arise to the right the truncus brachiocephalicus and to the left the a. subclavia sinistra (arteria) (Popesko et al., 1990; Fig. 1). The truncus brachiocephalicus, the a. carotis communis sinistra and the a. subclavia dextra as branches of the arcus aortae were described by Nejedlý (1965).

The truncus brachiocephalicus runs behind the cartilage of the first rib and the v. cava cranialis dextra on the left side of the trachea. Near the midline it courses to the right and then partly dorsally. Immediatelly after its origin gives off the a. carotis communis sinistra. The truncus brachiocephalicus is very short and divides into the a. carotis communis dextra and the a. subclavia dextra (Nejedlý, 1965). By other author the a. subclavia dextra was described as the first branch of the truncus brachiocephalicus (Popesko et al., 1990).

### Fig. 1. Basis cordis

7. a. subclavia sinistra; 8. truncus brachiocephalicus; 9. a. subclavia dextra; 10. truncus bicaroticus; 13. n. vagus dexter; 14. n. recurrens dexter; 15. n. recurrens sinister; 20. v. cava cranialis sinistra; 21. v. cava cranialis dextra; 22. v. cava caudalis; 23. v. azygos dextra; 24. truncus pulmonalis; 25. a. pulmonalis sinistra; 26. a. pulmonalis dextra; 27. vv. pulmonales; 28. atrium sinistrum; 29. atrium dextrum; 30. ventriculus sinister; 31. ventriculus dexter; 32. ligamentum arteriosum; 33. lymphonodi thoracici aortici; 34. a. bronchalis. v. Vena. Vv. Venae. n. Nervus. a. Arteria. Dorsal view (Popesko et al., 1990)

### **1.3.4 Arteria carotis communis**

In the neck region the a. carotis communis is covered by m. sternohyoideus (musculus), more cranially also by the m. sternothyroideus. It lies on the lateral surface of the trachea. The a. carotis communis lies dorsally to the esophagus, then laterally to the larynx. It reaches the maxillar angle laterally from the cranial end of the m. sternohyoideus and under the parotid gland. In this area it divides into the a. carotis interna and a. carotis externa (Nejedlý, 1965).

Variations in Origin of Arteries Supplying

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 69

cervical vertebrae cranially. It passes through the foramen tranversarium of the atlas, courses medially, then cranially and a little dorsally. It runs through the foramen vertebrale laterale of the atlas and gives off the a. spinalis dorsalis and ventralis. After this it penetrates the dura mater and continues to the cranial part of the medulla oblongata. On the caudal margin of the dorsal surface of the pars basilaris ossis occipitalis it is fused together with the contralateral a. vertebralis. This fusion forms the a. basilaris (Fig. 3). A. basilaris continues rostrally on the ventral surface of the medulla oblongata and pons. This artery gives off some branches which praticipate on the formation of the circle of Willis and by this way it

A. vertebralis dextra et sinistra located inside canalis transversarius of the cervical vertebrae.

a. A. cerebelli caudalis is the largest of the transverse branches arising from the a. basilaris on the ventral surface of the hindbrain. It originates about half way along the a. basilaris

b. A. cerebri caudalis is a paired vessel formed at the level of the rostral margin of the pons by the bifurcation of the a. basilaris. It passes at each side laterally and dorsally to the caudal portion of the cerebral hemisphere, giving secondary branches to the

c. A. cerebelli rostralis is a relatively large branch of the a. cerebri caudalis, arising near the origin of the latter and passing to the rostral portion of the cerebellum after giving

d. The end of the a. carotis interna lies on either side of the tuber cinereum. It turns forward, but is connected backwards with the a. cerebri caudalis by an a. communicans

e. A. cerebri media is given off from the a. carotis interna, branching over the middle

portion of the hemispere to supply most of its lateral and dorsal surfaces

The fusion of the bilateral aa. vertebrales to the a. basilaris. a. Arteria. Dorsal view

and passes laterally and up the side of the caudal part of the cerebellum

The arteries of the brain may be described on its ventral surface as follows:

participates on the arterial supplying of the brain (Nejedlý, 1965).

Fig. 3. Corrosion cast of the vertebral arteries

**1.3.8 The arteries of the brain**

diencephalon

caudalis

branches to the midbrain

Fig. 2. Scheme of the origin of the large arteries supplying the brain in the rabbit 1. arcus aortae; 2. truncus brachiocephalicus; 3. truncus bicaroticus; 4. a. subclavia dextra; 5. a. subclavia sinistra; 6. a. carotis communis dextra; 7. a. carotis communis sinistra; 8. a. vertebralis sinistra; 9. a. vertebralis dextra. a. Arteria. Lateral view

### **1.3.5 Arteria carotis interna**

The a. carotis interna as poorly developed artery it arises by the division of the a. carotis communis into the a. carotis externa and the a. carotis interna. From the a. carotis externa it is divided by m. styloglossus and m. stylopharyngeus. It is running dorsally on the medial side of the bulla tympanica ossis temporalis. It enters the canalis caroticus ossis temporalis and inside this canal continues into the skull cavity. It is running rostrally on the medial surface of the n. trigeminus (nervus) and on the lateral surface of the corpus ossis basisphenoidalis is directed into the sulcus caroticus. It turns dorsally on the medial surface on the place of the entrance of the n. oculomotorius into the fissura orbitalis. It crosses the n. oculomotorius to the right. In this way it makes three bends. After this it enters the ventral end of the canalis caroticus. Here it gives off the a. communicans caudalis and a. ophtalmica dorsalis and subsequently is divided into the a. carebri rostralis and a. cerebri media (Nejedlý, 1965).

### **1.3.6 Arteria subclavia**

The a. subclavia runs caudally and dorsally to the v. subclavia, above the n. cervicalis VIII., behind the origin of the m. sternomastoideus and m. pectoralis superficialis. The branches of a. subclavia are: truncus costocervicalis, a. vertebralis, a. cervicalis superficialis, a. mammaria interna, a. intercostalis suprema, a. cervicalis profunda and a. transversa colli. The direct continuation is a. axillaris (Nejedlý, 1965).

#### **1.3.7 Arteria vertebralis**

The a. vertebralis enters the foramen transversarium of the sixth cervical vertebra. It gives off rr. musculares (rami) and rr. spinales. It continues inside the canalis transversarius of the

Fig. 2. Scheme of the origin of the large arteries supplying the brain in the rabbit

vertebralis sinistra; 9. a. vertebralis dextra. a. Arteria. Lateral view

**1.3.5 Arteria carotis interna**

**1.3.6 Arteria subclavia**

**1.3.7 Arteria vertebralis**

The direct continuation is a. axillaris (Nejedlý, 1965).

1. arcus aortae; 2. truncus brachiocephalicus; 3. truncus bicaroticus; 4. a. subclavia dextra; 5. a. subclavia sinistra; 6. a. carotis communis dextra; 7. a. carotis communis sinistra; 8. a.

The a. carotis interna as poorly developed artery it arises by the division of the a. carotis communis into the a. carotis externa and the a. carotis interna. From the a. carotis externa it is divided by m. styloglossus and m. stylopharyngeus. It is running dorsally on the medial side of the bulla tympanica ossis temporalis. It enters the canalis caroticus ossis temporalis and inside this canal continues into the skull cavity. It is running rostrally on the medial surface of the n. trigeminus (nervus) and on the lateral surface of the corpus ossis basisphenoidalis is directed into the sulcus caroticus. It turns dorsally on the medial surface on the place of the entrance of the n. oculomotorius into the fissura orbitalis. It crosses the n. oculomotorius to the right. In this way it makes three bends. After this it enters the ventral end of the canalis caroticus. Here it gives off the a. communicans caudalis and a. ophtalmica dorsalis and subsequently is divided into the a. carebri rostralis and a. cerebri media (Nejedlý, 1965).

The a. subclavia runs caudally and dorsally to the v. subclavia, above the n. cervicalis VIII., behind the origin of the m. sternomastoideus and m. pectoralis superficialis. The branches of a. subclavia are: truncus costocervicalis, a. vertebralis, a. cervicalis superficialis, a. mammaria interna, a. intercostalis suprema, a. cervicalis profunda and a. transversa colli.

The a. vertebralis enters the foramen transversarium of the sixth cervical vertebra. It gives off rr. musculares (rami) and rr. spinales. It continues inside the canalis transversarius of the cervical vertebrae cranially. It passes through the foramen tranversarium of the atlas, courses medially, then cranially and a little dorsally. It runs through the foramen vertebrale laterale of the atlas and gives off the a. spinalis dorsalis and ventralis. After this it penetrates the dura mater and continues to the cranial part of the medulla oblongata. On the caudal margin of the dorsal surface of the pars basilaris ossis occipitalis it is fused together with the contralateral a. vertebralis. This fusion forms the a. basilaris (Fig. 3). A. basilaris continues rostrally on the ventral surface of the medulla oblongata and pons. This artery gives off some branches which praticipate on the formation of the circle of Willis and by this way it participates on the arterial supplying of the brain (Nejedlý, 1965).

Fig. 3. Corrosion cast of the vertebral arteries

A. vertebralis dextra et sinistra located inside canalis transversarius of the cervical vertebrae. The fusion of the bilateral aa. vertebrales to the a. basilaris. a. Arteria. Dorsal view

### **1.3.8 The arteries of the brain**

The arteries of the brain may be described on its ventral surface as follows:

Variations in Origin of Arteries Supplying

**1.4 Rabbit as experimental animal of the brain ischemia**

arteries supplying the brain with blood in the rabbit.

**2. Material and methods 2.1 Experimental animals**

ad libitum.

**2.2 Material**

**2.3 Methods**

**2.3.1 Surgical preparation of the rabbit**

rostrales.

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 71

The a. cerebri rostralis unites with that of the other side to form a short common trunk between the hemispheres, which redivides into the paired vessels supplying the medial surfaces. A complete anastomic loop is thus formed round the hypothalamus by the a. carotis interna, a. cerebri rostralis, a. communicans caudalis and a. cerebri caudalis. This is the circle of Willis or the circulus arteriosus cerebri (Popesko et al., 1990). Nejedlý (1965) described the a. communicans rostralis as a connection between the bilateral aa. cerebri

Over 20,000 rabbits were used for animal testing only in the UK in 2004.  This is most often carried out by ligation of major vessels, e.g. the truncus brachiocephalicus and the a. subclavia sinistra, in their place of origin (Hossmann 1998; Iwama et al., 2000; Pluta 1987). Harukuni and Bhardwaj (2006) reported ligation of the truncus brachiocephalicus and the a. subclavia sinistra as one way to induce total cerebral ischemia. Ischemia within the arteries branching from the vertebral arteries in the back of the brain may result in symptoms such as dizziness, vertigo, double vision, or weakness on both sides of the body. Other symptoms include, difficulty speaking, slurred speech and the loss of coordination (Beers et al., 2003). The aim of this study was to verify whether experimentally induced total cerebral ischemia in rabbits actually corresponds to total ischemia on the basis of the origin of certain vessels. We observed morphological variations in the origin and course of the

The study was carried out on 50 adult (age=140 days) New Zealand white rabbits (breed HY+), females (n=25) and males (n=25) of weight range 2.5-3 kg in an accredited experimental laboratory at the University of Veterinary Medicine in Kosice, Slovak Republic. The animals were kept in cages under standard conditions (temperature 15-20 °C, relative humidity 45 %, 12 hours light period) and fed granular mixed feed (O-10 NORM TYP, Spišské krmné zmesi, Spišské Vlachy, Slovak Republic). Drinking water was provided

The Batson΄s No. 17 Plastic Replica and Corrosion Kit (Polysciences Europe GmbH, Germany) was used as a casting medium. This consist of Base Solution A (2-Propenoic acid, 2-methyl-, 1,2-ethanediyl 1 ester, Dibutyl phthalate, Methyl methacrylate, Polymethyl methacrylate ), Catalyst (Acetone, Benzoyl peroxide, Dibutyl phthalate), Promoter C (Dibutyl 1 phthalate, N,N-Dimethyl-4-toluidine) and red pigment (1,2-Benzenedicarboxylic acid, bis[2-ethylhexyl ester], epoxidized soybean oil and 2-Naphthenecarboxylic acid).

The animals were injected intravenously with heparine (50, 000 UI/kg) 30 minutes before they were sacrified with intravenous injection of Embutramide (T-61, 0.3 mL/kg). The skin

Fig. 4. The arteries of the brain

1. bulbus olfactorius; 2 n. opticus; 3. n. oculomotorius; 4. n. trochlearis; 5. n. trigeminus; 6. n. abducens; 7. n. facialis; 8. n. vestibulocochlearis; 9. n. glossopharyngeus; 10. n. vagus; 11. n. accessorius; 12. n. hypoglossus; 18. corpus mamillare; 19. lobus piriformis; 20. chiasma opticum; 21. sulcus rhinalis lateralis; 24. paraflocculus; 26. a. carotis interna; 27., 28. a. cerebri media; 27. r. caudalis; 28. r. rostralis; 29. a. cerebri rostralis; 30. a. ethmoidalis interna; 31. a. communicans caudalis; 32. r. corporis mamillaris; 33. a. cerebri caudalis; 34. a. cerebelli rostralis; 35. rami ad pontem; 36. a. cerebelli caudalis; 37. a. basilaris; 38. a. vertebralis. a. Arteria. r. Ramus. Ventral View (Popesko et al., 1990)

f. A. cerebri rostralis is the continuation of the a. carotis interna after the origin of the a. cerebri media. It passes to the rostral portion of the ventral surface of the cerebral hemisphere and to the olfactory bulb.

The a. cerebri rostralis unites with that of the other side to form a short common trunk between the hemispheres, which redivides into the paired vessels supplying the medial surfaces. A complete anastomic loop is thus formed round the hypothalamus by the a. carotis interna, a. cerebri rostralis, a. communicans caudalis and a. cerebri caudalis. This is the circle of Willis or the circulus arteriosus cerebri (Popesko et al., 1990). Nejedlý (1965) described the a. communicans rostralis as a connection between the bilateral aa. cerebri rostrales.

### **1.4 Rabbit as experimental animal of the brain ischemia**

Over 20,000 rabbits were used for animal testing only in the UK in 2004.  This is most often carried out by ligation of major vessels, e.g. the truncus brachiocephalicus and the a. subclavia sinistra, in their place of origin (Hossmann 1998; Iwama et al., 2000; Pluta 1987). Harukuni and Bhardwaj (2006) reported ligation of the truncus brachiocephalicus and the a. subclavia sinistra as one way to induce total cerebral ischemia. Ischemia within the arteries branching from the vertebral arteries in the back of the brain may result in symptoms such as dizziness, vertigo, double vision, or weakness on both sides of the body. Other symptoms include, difficulty speaking, slurred speech and the loss of coordination (Beers et al., 2003). The aim of this study was to verify whether experimentally induced total cerebral ischemia in rabbits actually corresponds to total ischemia on the basis of the origin of certain vessels. We observed morphological variations in the origin and course of the arteries supplying the brain with blood in the rabbit.

### **2. Material and methods**

### **2.1 Experimental animals**

The study was carried out on 50 adult (age=140 days) New Zealand white rabbits (breed HY+), females (n=25) and males (n=25) of weight range 2.5-3 kg in an accredited experimental laboratory at the University of Veterinary Medicine in Kosice, Slovak Republic. The animals were kept in cages under standard conditions (temperature 15-20 °C, relative humidity 45 %, 12 hours light period) and fed granular mixed feed (O-10 NORM TYP, Spišské krmné zmesi, Spišské Vlachy, Slovak Republic). Drinking water was provided ad libitum.

### **2.2 Material**

70 Advances in the Preclinical Study of Ischemic Stroke

1. bulbus olfactorius; 2 n. opticus; 3. n. oculomotorius; 4. n. trochlearis; 5. n. trigeminus; 6. n. abducens; 7. n. facialis; 8. n. vestibulocochlearis; 9. n. glossopharyngeus; 10. n. vagus; 11. n. accessorius; 12. n. hypoglossus; 18. corpus mamillare; 19. lobus piriformis; 20. chiasma opticum; 21. sulcus rhinalis lateralis; 24. paraflocculus; 26. a. carotis interna; 27., 28. a. cerebri media; 27. r. caudalis; 28. r. rostralis; 29. a. cerebri rostralis; 30. a. ethmoidalis interna; 31. a. communicans caudalis; 32. r. corporis mamillaris; 33. a. cerebri caudalis; 34. a. cerebelli rostralis; 35. rami ad pontem; 36. a. cerebelli caudalis; 37. a. basilaris; 38. a. vertebralis. a.

f. A. cerebri rostralis is the continuation of the a. carotis interna after the origin of the a. cerebri media. It passes to the rostral portion of the ventral surface of the cerebral

Fig. 4. The arteries of the brain

Arteria. r. Ramus. Ventral View (Popesko et al., 1990)

hemisphere and to the olfactory bulb.

The Batson΄s No. 17 Plastic Replica and Corrosion Kit (Polysciences Europe GmbH, Germany) was used as a casting medium. This consist of Base Solution A (2-Propenoic acid, 2-methyl-, 1,2-ethanediyl 1 ester, Dibutyl phthalate, Methyl methacrylate, Polymethyl methacrylate ), Catalyst (Acetone, Benzoyl peroxide, Dibutyl phthalate), Promoter C (Dibutyl 1 phthalate, N,N-Dimethyl-4-toluidine) and red pigment (1,2-Benzenedicarboxylic acid, bis[2-ethylhexyl ester], epoxidized soybean oil and 2-Naphthenecarboxylic acid).

### **2.3 Methods**

### **2.3.1 Surgical preparation of the rabbit**

The animals were injected intravenously with heparine (50, 000 UI/kg) 30 minutes before they were sacrified with intravenous injection of Embutramide (T-61, 0.3 mL/kg). The skin

Variations in Origin of Arteries Supplying

Fig. 5. Aortic arch of the rabbit without variations

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 73

a. subclavia dextra by terminal division of truncus brachiocephalicus (Fig. 5). The a. carotis

Fig. 6. Aortic arch of the rabbit with variation in origin of a. carotis communis sinistra

communis sinistra originated from arcus aortae in 6 % (3 animals; Fig. 6).

was subsequently removed as far as possible to prevent it from sticking to the corrosive cast in the maceration process. The thoracic cavity was opened from the left side by removing of the ribs. After the opening of the pericardial cavity a ligature was introduced to the ascending aorta. The aorta was cannulated through the left ventricle. The perfusion started after the fixation of the cannula in the ascending aorta with the ligature. The right vestibule was opened to lower the pressure in the vessels to ensure good injection. The vascular network was manually perfused through the fixed cannula in the ascending aorta for approximately 15-20 minutes with 2.5-3 l of warm (37 °C), 0.9 % NaOH in 0.01 M phosphate, pH 7.3 (Hossler & Monson, 1995).

### **2.3.2 Preparation of the casting medium**

The red pigment was added to the Base solution A prior to mixing the catalyst and promoter. The pigment was added in the amount of 5 %. It was mixed and divided into two equal parts (each part=25 mL). To the first half Catalyst in amount of 12 mL was added and mixed. To the second half Promoter C in amount of 12 drops was added and mixed. Then these two parts were mixed together.

### **2.3.3 Application of the casting medium**

The arterial network was filled with the casting medium manually through the same cannula inserted in the ascending aorta. Adequate filling was determined by the visualization of an even distribution of the casting medium (red) throughout the superficial vessels of the body. After the vascular casting is complete, the animals must not be manipulated for at least 30 minutes and then must be submersed in water at a temperature ranging from 40 °C to 60 °C for a period of 24 hours for full polymerization of the casting medium (Lametschwandtner et al., 1990).

### **2.3.4 Corrosion**

The corrosion as the dissolution of tissues surrounding the cast was performed by potassium hydroxide (KOH) at the concentration of solution 2–4 % for a period of 2 days. For the corrosion to be faster, the solution must remain at a constant temperature of 40 °C (Lametschwandtner et al., 1990). The solution for the corrosion was changed every 12 hours. After the surrounding tissue was dissolved vascular castings were rinsed in running water for removing the rests of the soft tissues. Specimens were dried at the room temperature by air exposure (Flešárová et al., 2003).

### **3. Results and discussion**

### **3.1 Variations in origin**

### **3.1.1 A. carotis communis**

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus and the a. subclavia sinistra from the arcus aortae in 92 % of cases (46 animals). a. Arteria. Ventral view. Macroscopic image

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus, the a. subclavia sinistra and the a. carotis communis sinistra from the arcus aortae in 6 % (3 animals). a. Arteria. Ventral view. Macroscopic image

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as the first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the

was subsequently removed as far as possible to prevent it from sticking to the corrosive cast in the maceration process. The thoracic cavity was opened from the left side by removing of the ribs. After the opening of the pericardial cavity a ligature was introduced to the ascending aorta. The aorta was cannulated through the left ventricle. The perfusion started after the fixation of the cannula in the ascending aorta with the ligature. The right vestibule was opened to lower the pressure in the vessels to ensure good injection. The vascular network was manually perfused through the fixed cannula in the ascending aorta for approximately 15-20 minutes with 2.5-3 l of warm (37 °C), 0.9 % NaOH in 0.01 M phosphate,

The red pigment was added to the Base solution A prior to mixing the catalyst and promoter. The pigment was added in the amount of 5 %. It was mixed and divided into two equal parts (each part=25 mL). To the first half Catalyst in amount of 12 mL was added and mixed. To the second half Promoter C in amount of 12 drops was added and mixed. Then

The arterial network was filled with the casting medium manually through the same cannula inserted in the ascending aorta. Adequate filling was determined by the visualization of an even distribution of the casting medium (red) throughout the superficial vessels of the body. After the vascular casting is complete, the animals must not be manipulated for at least 30 minutes and then must be submersed in water at a temperature ranging from 40 °C to 60 °C for a period of 24 hours for full polymerization of the casting

The corrosion as the dissolution of tissues surrounding the cast was performed by potassium hydroxide (KOH) at the concentration of solution 2–4 % for a period of 2 days. For the corrosion to be faster, the solution must remain at a constant temperature of 40 °C (Lametschwandtner et al., 1990). The solution for the corrosion was changed every 12 hours. After the surrounding tissue was dissolved vascular castings were rinsed in running water for removing the rests of the soft tissues. Specimens were dried at the room temperature by

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus and the a. subclavia sinistra from the arcus aortae in 92 % of cases (46

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus, the a. subclavia sinistra and the a. carotis communis sinistra from the

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as the first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the

arcus aortae in 6 % (3 animals). a. Arteria. Ventral view. Macroscopic image

pH 7.3 (Hossler & Monson, 1995).

**2.3.2 Preparation of the casting medium**

these two parts were mixed together.

**2.3.3 Application of the casting medium**

medium (Lametschwandtner et al., 1990).

air exposure (Flešárová et al., 2003).

animals). a. Arteria. Ventral view. Macroscopic image

**3. Results and discussion**

**3.1 Variations in origin 3.1.1 A. carotis communis**

**2.3.4 Corrosion**

a. subclavia dextra by terminal division of truncus brachiocephalicus (Fig. 5). The a. carotis communis sinistra originated from arcus aortae in 6 % (3 animals; Fig. 6).

Fig. 5. Aortic arch of the rabbit without variations

Fig. 6. Aortic arch of the rabbit with variation in origin of a. carotis communis sinistra

Variations in Origin of Arteries Supplying

Fig. 8. A. vertebralis with its typical origin

Fig. 9. Atypical origin of the a. vertebralis

Macroscopic image

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 75

Vascular corrosion cast displaying the origin of the a. vertebralis dextra et sinistra from the a. subclavia dextra et sinistra in 86 % of cases (43 animals). a. Arteria. Ventral view.

Vascular corrosion cast displaying the origin of the a. vertebralis sinistra from the arcus

aortae in 10 % of cases (5 animals). a. Arteria. Dorsal view. Macroscopic image

### **3.1.2 A. subclavia**

In 98 % of cases (49 animals) the a. subclavia sinistra originated from the arcus aortae (Fig. 5). In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra (Fig. 6).

Fig. 7. Aortic arch of the rabbit with variation in origin of a. subclavia dextra

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra from the arcus aortae in 2 % (1 animal). a. Arteria. Dorsolateral view. Macroscopic image

### **3.1.3 A. vertebralis**

In 86 % of cases (43 animals) the a. vertebralis sinistra originated directly from the a. subclavia sinistra (Fig. 8) in 10 % of cases (5 animals) it originated from the arcus aortae as an independent branch (Fig. 9) and in 4 % of cases (2 animals) it arose from the arcus aortae as a common trunk with the a. scapularis descendens. The a. vertebralis dextra originated from the a. subclavia dextra in 98 % (49 animals) of cases. In that case we observed two aa. vertebralis dextrae (arteriae) with two different origins.

The a. vertebralis dextra I originated from the a. subclavia dextra and the a. vertebralis dextra II arose from the common trunk with the a. cervicalis superficialis dextra that originated from the a. carotis communis dextra.

After a short distance, they merged between the fifth and sixth cervical vertebrae into a single a. vertebralis dextra, which then entered the canalis transversarius at the level of the fifth cervical vertebra (Fig. 10). In summary, the origin of both aa. vertebrales varied in 16 % of cases (8 animals). In 8 % (4 animals) we found a bypass between the a. vertebralis sinistra and the a. basilaris (Fig. 11). This a. basilaris was also formed by the fusion of the a. vertebralis dextra et sinistra.

In 98 % of cases (49 animals) the a. subclavia sinistra originated from the arcus aortae (Fig. 5). In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus, the a. subclavia

Fig. 7. Aortic arch of the rabbit with variation in origin of a. subclavia dextra

(1 animal). a. Arteria. Dorsolateral view. Macroscopic image

vertebralis dextrae (arteriae) with two different origins.

originated from the a. carotis communis dextra.

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra from the arcus aortae in 2 %

In 86 % of cases (43 animals) the a. vertebralis sinistra originated directly from the a. subclavia sinistra (Fig. 8) in 10 % of cases (5 animals) it originated from the arcus aortae as an independent branch (Fig. 9) and in 4 % of cases (2 animals) it arose from the arcus aortae as a common trunk with the a. scapularis descendens. The a. vertebralis dextra originated from the a. subclavia dextra in 98 % (49 animals) of cases. In that case we observed two aa.

The a. vertebralis dextra I originated from the a. subclavia dextra and the a. vertebralis dextra II arose from the common trunk with the a. cervicalis superficialis dextra that

After a short distance, they merged between the fifth and sixth cervical vertebrae into a single a. vertebralis dextra, which then entered the canalis transversarius at the level of the fifth cervical vertebra (Fig. 10). In summary, the origin of both aa. vertebrales varied in 16 % of cases (8 animals). In 8 % (4 animals) we found a bypass between the a. vertebralis sinistra and the a. basilaris (Fig. 11). This a. basilaris was also formed by the fusion of the a.

**3.1.2 A. subclavia**

**3.1.3 A. vertebralis**

vertebralis dextra et sinistra.

dextra and the a. subclavia sinistra (Fig. 6).

Fig. 8. A. vertebralis with its typical origin

Vascular corrosion cast displaying the origin of the a. vertebralis dextra et sinistra from the a. subclavia dextra et sinistra in 86 % of cases (43 animals). a. Arteria. Ventral view. Macroscopic image

Fig. 9. Atypical origin of the a. vertebralis

Vascular corrosion cast displaying the origin of the a. vertebralis sinistra from the arcus aortae in 10 % of cases (5 animals). a. Arteria. Dorsal view. Macroscopic image

Variations in Origin of Arteries Supplying

Fig. 12. Typical origin of a. carotis interna

Fig. 13. Atypical origin of a. carotis interna

caroticus. a. Arteria. Ventrolateral view. Macroscopic image

**3.1.4 A. carotis interna**

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 77

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna by the terminal division of the a. carotis communis together with the a. carotis externa in 94 % (47 animals). Note the entrance of the a. carotis interna to the canalis

### Fig. 10. Doubled a. vertebralis

Vascular corrosion cast displaying two aa. vertebrales dextrae. Note merging of the two arteriae into a single vessel. a. Arteria. aa. Arteriae. Lateral view. Macroscopic image

Fig. 11. Variation in formation of a. basilaris

Vascular corrosion cast displaying the bypass between the a. vertebralis sinistra and a. basilaris in 8 % (4 animals). a. Arteria. Dorsal view. Macroscopic image

### **3.1.4 A. carotis interna**

76 Advances in the Preclinical Study of Ischemic Stroke

Vascular corrosion cast displaying two aa. vertebrales dextrae. Note merging of the two

Vascular corrosion cast displaying the bypass between the a. vertebralis sinistra and a.

basilaris in 8 % (4 animals). a. Arteria. Dorsal view. Macroscopic image

arteriae into a single vessel. a. Arteria. aa. Arteriae. Lateral view. Macroscopic image

Fig. 10. Doubled a. vertebralis

Fig. 11. Variation in formation of a. basilaris

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna by the terminal division of the a. carotis communis together with the a. carotis externa in 94 % (47 animals). Note the entrance of the a. carotis interna to the canalis caroticus. a. Arteria. Ventrolateral view. Macroscopic image

Fig. 12. Typical origin of a. carotis interna

Fig. 13. Atypical origin of a. carotis interna

Variations in Origin of Arteries Supplying

Fig. 15. Variation in arrangement of a. cerebelli caudalis

dextra et sinistra as independent branches of the arcus aortae.

**3.2 Discussion**

**3.2.2 A. subclavia**

**3.2.1 A. carotis communis**

Vascular corrosion cast. The a. cerebelli rostralis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %). Dorsal view. Macroscopic image

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the a. subclavia dextra by the terminal division of the truncus brachiocephalicus. Popesko et al. (1990) described a. carotis communis dextra et sinistra as branches arising from truncus bicaroticus by its terminal division. Truncus bicaroticus was described as the direct continuation of the truncus brachiocephalicus. The a. carotis communis sinistra as branch of the arcus aortae in 4 % (2 animals) was also described by Ding (2006). By Nejedlý (1965) the origin of the a. carotis communis sinistra from the arcus aortae was described as a typical arrangement of branches of the arcus aortae. In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus. Nellie (1930) described this arrangement in all studied animals. White (1893) by studying 700 rabbits found in one case the a. carotis communis

In 98 % (49 animals) the a. subclavia dextra arise together with the a. carotis communis dextra by the terminal division of the truncus brachiocephalicus. By some authors the a.

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 79

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna from the common trunk with the a. occipitalis in 6 % (3 animals). a. Arteria. Caudoventral view. Macroscopic image

In 94 % (47 animals) the a. carotis interna arised by terminal division of the a. carotis communis together with the a. carotis externa (Fig. 12). In 6 % (3 animals) the a. carotis interna originated from a common trunk with the a. occipitalis (Fig. 13). The trunk is a branch of the a. carotis communis.

### **3.1.5 The arteries of the brain**

In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level (Fig. 14). In 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra (Fig. 15). In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra.

The a. cerebri caudalis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %).

The a. cerebri media dextra et sinistra originated from the a. carotis interna at the same level in all studied animals (100 %).

Fig. 14. Typical arrangement of a. cerebelli caudalis

Vascular corrosion cast. In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level from the a. basilaris. Dorsal view. Macroscopic image

The a. cerebri rostralis dextra et sinistra as the direct continuation from the a. carotis interna originated at the same level in all studied animals (100 %).

These all arteries were divided into the r. rostralis and r. caudalis. In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r. rostralis, r. medius and r. caudalis.

Fig. 15. Variation in arrangement of a. cerebelli caudalis

Vascular corrosion cast. The a. cerebelli rostralis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %). Dorsal view. Macroscopic image

### **3.2 Discussion**

78 Advances in the Preclinical Study of Ischemic Stroke

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna from the common trunk with the a. occipitalis in 6 % (3 animals). a. Arteria.

In 94 % (47 animals) the a. carotis interna arised by terminal division of the a. carotis communis together with the a. carotis externa (Fig. 12). In 6 % (3 animals) the a. carotis interna originated from a common trunk with the a. occipitalis (Fig. 13). The trunk is

In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level (Fig. 14). In 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra (Fig. 15). In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra. The a. cerebri caudalis dextra et sinistra originated from the a. basilaris at the same level in

The a. cerebri media dextra et sinistra originated from the a. carotis interna at the same level

Caudoventral view. Macroscopic image

a branch of the a. carotis communis.

**3.1.5 The arteries of the brain**

all studied animals (100 %).

in all studied animals (100 %).

Fig. 14. Typical arrangement of a. cerebelli caudalis

originated at the same level in all studied animals (100 %).

Vascular corrosion cast. In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level from the a. basilaris. Dorsal view. Macroscopic image

cerebri caudalis sinistra was divided into the r. rostralis, r. medius and r. caudalis.

The a. cerebri rostralis dextra et sinistra as the direct continuation from the a. carotis interna

These all arteries were divided into the r. rostralis and r. caudalis. In 10 % (5 animals) the a.

### **3.2.1 A. carotis communis**

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the a. subclavia dextra by the terminal division of the truncus brachiocephalicus. Popesko et al. (1990) described a. carotis communis dextra et sinistra as branches arising from truncus bicaroticus by its terminal division. Truncus bicaroticus was described as the direct continuation of the truncus brachiocephalicus. The a. carotis communis sinistra as branch of the arcus aortae in 4 % (2 animals) was also described by Ding (2006). By Nejedlý (1965) the origin of the a. carotis communis sinistra from the arcus aortae was described as a typical arrangement of branches of the arcus aortae. In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus. Nellie (1930) described this arrangement in all studied animals. White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra as independent branches of the arcus aortae.

### **3.2.2 A. subclavia**

In 98 % (49 animals) the a. subclavia dextra arise together with the a. carotis communis dextra by the terminal division of the truncus brachiocephalicus. By some authors the a.

Variations in Origin of Arteries Supplying

occurrence of found variations.

also in a smaller number of animals used in experiments.

No. 3, pp. 129-131, ISSN 0022-4804

978-0-230-60014-0, Baltimore, USA

Oxford, USA

8865

116, ISSN 0001-7213

possible variations.

**5. References**

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 81

The variations in the origin of arteries supplying the brain which we found point to the possibility of induction only a partial brain ischemia in a given set of animals. The probability of causing the partial brain ischemia may be the same as the percentage of the

One way to avoid obtaining of distorted results is the ligation of the arteries before their entering to the target organ, in this case to the brain. These arteries can be the a. basilaris or the a. carotis interna. The best results probably would have been achieved by ligation of the arteries on the ventral surface of the brain that are directly involved in the blood supply of the nerve tissue. However, this method is time consuming and surgically very difficult in. Another possibility is the detailed preparation of arteries in the place of their origin to avoid

With this work we tried to emphasize the need for more detailed knowledge of the circulatory system of the rabbit, which is one of the ways to achieve more objective results

Beers, M.; Fletcher, A.; Jones, T. & Porter, R. (2003). *The Merck Manual of Medical Information*.

Burkholz, H. (1997). Giving Thalidomide a Second Chance. In: *FDA Consumer* (US

Cantu, R.C. & Hegsted, D.M. (1970). Comparison of cerebrovascular perfusion after

Carbone, L. (2004). *What Animals Want*. Oxford University Press. ISBN<PHONE_NUMBER>960,

Cohen, B.J. & Loew F.M. (1984). *Laboratory Animal Medicine: Historical Perspectives in Laboratory Animal Medicine*. (eds J.G. Fox, B.J. Cohen and F.M. Loew) Academic

Cohn, M. (2010). *Alternatives to Animal Testing Gaining Ground*. The Baltimore Sun. ISBN

Ding, Y.H.; Dai, D.; Layton, K.F.; Lewis, D.A.; Danielson, M.A.; Kadirvel, R.; Cloft, H.J. &

Flešárová, S.; Tesfaye, A.; Danko J, Bátor, R.; Dorko, F. & Barta, A. (2003). Morphological

Harukuni, I. & Bhardwaj, A. (2006). Mechanisms of Brain Injury after Global Cerebral

Hossler, F.E. & Monson, F.C. (1995). Microvasculature of the rabbit urinary bladder. *The*

Hossmann, K-A. (1998). Experimental models for the investigation of brain ischemia.

Ishiyama, T.; Shibuya, K.; Ichikawa, M.; Masamune, T.; Kiuchi, R.; Sessler, D.I. &

Matsukawa, T. (2010). Cerebral pial vascular changes under propofol or

Ischemia. *Neurological Clinic*, Vol.24, No.1, pp. 1-21, ISSN 0733-8619

 *Anatomical Record*, Vol.243, No.4, pp. 438-448, ISSN 1932-8486

*Cardiovascular Research*, Vol.39, No.1, pp. 106-120, ISSN 0008-6363

Kallmes, D.F. (2006). Vascular Anatomic Variation in Rabbits. *Journal of Vascular and Interventional Radiobiology*, Vol.17, No.6, pp. 1031-1035, ISSN 0345-

variations of arcus aortae in rabbits. *Acta Veterinaria Brno*, Vol. 72, No. 7, pp.

ischemia in normal and atherosclerotic rabbits. *Journal of Surgical Research*, Vol. 10,

New York: Merck & Co. Inc., ISBN<PHONE_NUMBER>, New York, USA

 Food and Drug Administration). 06 Jun 2011. Available from: http://findarticles.com/p/articles/mi\_m1370/is\_n6\_v31/ai\_19903511/

Press, Inc., ISBN<PHONE_NUMBER>, Orlando, Florida, USA

subclavia dextra was described as the first branch arising from the truncus brachiocephalicus (Popesko et al., 1990). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases from the arcus aortae and Nejedlý (1965) described it as typical arrangement of the branches of the arcus aortae. White (1893) by studying 700 pieces rabbits found in one case a. subclavia dextra et sinistra as independent branches of the arcus aortae.

### **3.2.3 A. vertebralis**

Until now the scientific literature has cited almost exclusively the uniform origin of the a. vertebralis from the a. subclavia (Nejedlý, 1965; Popesko et al., 1990). It was not described independent origin from the arcus aortae or the doubled a. vertebralis like in our cases. In 8 % (4 animals) we found the bypass between the a. vertebralis sinistra and the a. basilaris at the place of fusion of the a. vertebralis dextra et sinistra to the a. basilaris.

### **3.2.4 A. carotis interna**

We found the origin of the a. carotis interna and the a. carotis externa by the terminal division of the a. carotis communis in 92 % (46 animals). The same origin was described by Nejedlý (1965) and Popesko et al. (1990). But we found except this typical arrangement the origin of the a. carotis interna from the a. occipitalis in 8 % (4 animals).

### **3.2.5 The arteries of the brain**

Until now the origin of the bilateral a. cerebri rostralis et caudalis and a. cerebelli rostralis et caudalis was described at the same level from the a. basilaris and the a. carotis interna (Nejedlý, 1965; Popesko et al., 1990). We found that in 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra. In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra.

These all arteries were divided into the r. rostralis and r. caudalis (Nejedlý, 1965; Popesko et al., 1990). In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r. rostralis, r. medius and r. caudalis.

The bilateral aa. cerebri rostrales are fused together. The same arrangement was described by Popesko et al. (1990). The a. communicans rostralis as a connection between bilateral aa. cerebri rostrales was described by Nejedlý (1965).

### **4. Conclusion**

The effect of various chemical substances (Cantu **&** Hegsted**, 1970**) on the brain nerve tissue damaged by ischemia as well as various pathological and pathophysiological changes induced by the total cerebral ischemia in rabbits and other laboratory animals are the subject of many studies (Ishiyama et al., 2010).

The place of origin of the truncus brachiocephalicus and the a. subclavia sinistra are most commonly used to induce the total cerebral ischemia by ligation (Hossmann 1998; Iwama et al., 2000; Pluta, 1987). Harukuni and Bhardwaj (2006) present also the ligation of the truncus brachiocephalicus and the a. subclavia sinistra as a possible way to induce the total cerebral ischemia. The question is, whether this method of induction of the total cerebral ischemia is correct.

The variations in the origin of arteries supplying the brain which we found point to the possibility of induction only a partial brain ischemia in a given set of animals. The probability of causing the partial brain ischemia may be the same as the percentage of the occurrence of found variations.

One way to avoid obtaining of distorted results is the ligation of the arteries before their entering to the target organ, in this case to the brain. These arteries can be the a. basilaris or the a. carotis interna. The best results probably would have been achieved by ligation of the arteries on the ventral surface of the brain that are directly involved in the blood supply of the nerve tissue. However, this method is time consuming and surgically very difficult in. Another possibility is the detailed preparation of arteries in the place of their origin to avoid possible variations.

With this work we tried to emphasize the need for more detailed knowledge of the circulatory system of the rabbit, which is one of the ways to achieve more objective results also in a smaller number of animals used in experiments.

### **5. References**

80 Advances in the Preclinical Study of Ischemic Stroke

subclavia dextra was described as the first branch arising from the truncus brachiocephalicus (Popesko et al., 1990). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases from the arcus aortae and Nejedlý (1965) described it as typical arrangement of the branches of the arcus aortae. White (1893) by studying 700 pieces rabbits found in one case a. subclavia dextra et sinistra as

Until now the scientific literature has cited almost exclusively the uniform origin of the a. vertebralis from the a. subclavia (Nejedlý, 1965; Popesko et al., 1990). It was not described independent origin from the arcus aortae or the doubled a. vertebralis like in our cases. In 8 % (4 animals) we found the bypass between the a. vertebralis sinistra and the a. basilaris at

We found the origin of the a. carotis interna and the a. carotis externa by the terminal division of the a. carotis communis in 92 % (46 animals). The same origin was described by Nejedlý (1965) and Popesko et al. (1990). But we found except this typical arrangement the

Until now the origin of the bilateral a. cerebri rostralis et caudalis and a. cerebelli rostralis et caudalis was described at the same level from the a. basilaris and the a. carotis interna (Nejedlý, 1965; Popesko et al., 1990). We found that in 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra. In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than

These all arteries were divided into the r. rostralis and r. caudalis (Nejedlý, 1965; Popesko et al., 1990). In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r. rostralis,

The bilateral aa. cerebri rostrales are fused together. The same arrangement was described by Popesko et al. (1990). The a. communicans rostralis as a connection between bilateral aa.

The effect of various chemical substances (Cantu **&** Hegsted**, 1970**) on the brain nerve tissue damaged by ischemia as well as various pathological and pathophysiological changes induced by the total cerebral ischemia in rabbits and other laboratory animals are the subject

The place of origin of the truncus brachiocephalicus and the a. subclavia sinistra are most commonly used to induce the total cerebral ischemia by ligation (Hossmann 1998; Iwama et al., 2000; Pluta, 1987). Harukuni and Bhardwaj (2006) present also the ligation of the truncus brachiocephalicus and the a. subclavia sinistra as a possible way to induce the total cerebral ischemia. The question is, whether this method of induction of the total cerebral ischemia is

the place of fusion of the a. vertebralis dextra et sinistra to the a. basilaris.

origin of the a. carotis interna from the a. occipitalis in 8 % (4 animals).

independent branches of the arcus aortae.

**3.2.3 A. vertebralis**

**3.2.4 A. carotis interna**

**3.2.5 The arteries of the brain**

the a. cerebelli caudalis dextra.

cerebri rostrales was described by Nejedlý (1965).

of many studies (Ishiyama et al., 2010).

r. medius and r. caudalis.

**4. Conclusion**

correct.

**Part 2**

**Pathophysiology of Ischemic**

**or Anoxic Damage**

 sevoflurane anesthesia during global cerebral ischemia and reperfusion in rabbits. *Journal of neurosurgical anesthesiology*, Vol. 22, No. 3, pp. 207-213, ISSN 0898-4921

## **Part 2**

**Pathophysiology of Ischemic or Anoxic Damage**

82 Advances in the Preclinical Study of Ischemic Stroke

Iwama, H.; Akama, Y. & Tase, C. (2000). Global brain ischemia produced by clamping left subclavian artery and bicarotid trunk in the rabbit. *American Journal of Emergency*

Lametschwandtner, A.; Lametschwandtner, U. & Weiger, T. (1990). Scanning electron

Nellie, B.E. (1930). Note on an abnormality in the blood vascular system of the rabbit.

Pluta, R. (1987). Resuscitation of the rabbit brain after acute complete ischemia lasting up

Popesko, P.; Rajtová, V. & Horák, J. (1990). *Anatomic atlas of small laboratory animals.*

Tolwani, R.J.; Jakowec, M.W.; Petzinger, G.M.; Green, S. & Waggie, K. (1999).

Vespa, P.; Bergsneider, M.; Hattori, N.; Wu, H-M.; Huang, S-CH.; Martin, N.A.; Glenn,

White, P.J. (1893). Unusual Origin of Arteries in the Rabbit. *Nature*, Vol.47, No.1216, pp.

Wilmut, I.; Schnieke, A.E.; McWhir, J.; Kind, A.J. & Campbell, K.H. (1997). Viable

*Laboratory animal science,* Vol.49, No.4, pp. 363–371, ISSN 0023-6772

 review. *Scanning microscopy*, Vol.4, No.4, pp. 889-941, ISSN 0891-7035 Nejedlý, K. (1965). *Biology and systematic anatomy of laboratory animals,* SPN, ISBN 16-924- 65,

*Journal of Anatomy,* Vol.65, No.1*,* pp. 152-153, ISSN 0021-8782

*Resuscitation,* Vol.15, No.4, pp. 267-287, ISSN 0300-95721987

Príroda, ISBN 80-07-00040-2, Bratislava, Slovak Republic

Vol.25, No.6, pp. 763–774, ISSN 0271-678X

No.6619, pp. 810–813, ISSN 0028-0836

*Medicine*, Vol.18, No.1, pp. 31-35, ISSN 0263-1933

ISSN 0898-4921

Praha, Czech Republic

365, ISSN 0028-0836

 sevoflurane anesthesia during global cerebral ischemia and reperfusion in rabbits. *Journal of neurosurgical anesthesiology*, Vol. 22, No. 3, pp. 207-213,

microscopy of vascular corrosion casts.technique and aplications: uptade

to one hour: pathophysiological and pathomorphological observations.

Experimental models of Parkinson's disease: insights from many models.

 T.C.; McArthur, D.L. & Hovda, D.A. (2005). Metabolic crisis without brain ischemia is common after traumatic brain injury: a combined microdialysis and positron emission tomography study. *Journal of Cerebral Blood Flow & Metabolism,*

offspring derived from fetal and adult mammalian cell. *Nature,* Vol.385,

**5**

*Canada*

**Cerebral Ischemia Induced Proteomic**

Willard J. Costain1, Arsalan S. Haqqani2, Ingrid Rasquinha1,

*1Glycosyltransferases and Neuroglycomics, Institute for Biological Sciences,*

*2Proteomics, Institute for Biological Sciences, National Research Council, Ottawa, ON,*

The synapse is the focal point for neuronal communication and neuron-glia interactions. Synaptic structure and function are intimately related and many of the proteins that provide structure to the synapse also regulate synaptic function (Abe et al. 2004, Couchman 2003, Ehlers 2002, Passafaro et al. 2003). The synaptic structure - function relationship is highly apparent during pathological conditions. This is exemplified in neurodegenerative disorders, such as Alzheimer's disease, where synaptic function is positively correlated with neuronal function (Gasic & Nicotera 2003) and viability (Deisseroth et al. 2003), with

Maintenance of synaptic structure and functionality is a process that is highly energy dependent. Studies of synaptosomal morphology and metabolism have indicated that the synapse is highly susceptible to ischemic damage (Pastuszko et al. 1982, Rafalowska et al. 1980, Sulkowski et al. 2002, Enright et al. 2007, Zhang & Murphy 2007). This exceptional requirement for energy necessitates the localization of numerous mitochondria proximal to the synaptic bouton and mitochondria are a commonly observed feature in synaptosomal preparations (Costain et al. 2008). Synaptosomal metabolic activity (Rafalowska et al. 1980) and the rate of neurotransmitter re-uptake (Pastuszko et al. 1982) are decreased following acute hypoxia. Furthermore, Sulkowski *et al* (2002) observed that ischemia decreases

synaptosomal oxygen consumption and metabolic capacity for a period of 24 hours.

Cerebral ischemia induces a marked depletion in synaptic vesicle content and increases the number of damaged mitochondria (Sulkowski et al. 2002, Costain et al. 2008). Importantly, ischemia-induced alterations in synaptosomal morphology are indistinguishable from that of brain slices (Sulkowski et al. 2002). Ischemia-like conditions (hypoxic stress or excitotoxicity) induce dramatic and rapidly reversible structural/morphological changes in dendritic spines (Hasbani et al. 2001, Mattson et al. 1998, Park et al. 1996, Enright et al. 2007,

synaptic pathology preceding cell death (Gasic & Nicotera 2003).

**1. Introduction**

**Alterations: Consequences for**

**the Synapse and Organelles**

Marie-Soleil Giguere2 and Jacqueline Slinn3

*3Cerebrovascular Research, Institute for Biological Sciences,*

*National Research Council, Ottawa, ON,*

*National Research Council, Ottawa, ON,*

Willard J. Costain1, Arsalan S. Haqqani2, Ingrid Rasquinha1, Marie-Soleil Giguere2 and Jacqueline Slinn3 *1Glycosyltransferases and Neuroglycomics, Institute for Biological Sciences, National Research Council, Ottawa, ON, 2Proteomics, Institute for Biological Sciences, National Research Council, Ottawa, ON, 3Cerebrovascular Research, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada*

### **1. Introduction**

The synapse is the focal point for neuronal communication and neuron-glia interactions. Synaptic structure and function are intimately related and many of the proteins that provide structure to the synapse also regulate synaptic function (Abe et al. 2004, Couchman 2003, Ehlers 2002, Passafaro et al. 2003). The synaptic structure - function relationship is highly apparent during pathological conditions. This is exemplified in neurodegenerative disorders, such as Alzheimer's disease, where synaptic function is positively correlated with neuronal function (Gasic & Nicotera 2003) and viability (Deisseroth et al. 2003), with synaptic pathology preceding cell death (Gasic & Nicotera 2003).

Maintenance of synaptic structure and functionality is a process that is highly energy dependent. Studies of synaptosomal morphology and metabolism have indicated that the synapse is highly susceptible to ischemic damage (Pastuszko et al. 1982, Rafalowska et al. 1980, Sulkowski et al. 2002, Enright et al. 2007, Zhang & Murphy 2007). This exceptional requirement for energy necessitates the localization of numerous mitochondria proximal to the synaptic bouton and mitochondria are a commonly observed feature in synaptosomal preparations (Costain et al. 2008). Synaptosomal metabolic activity (Rafalowska et al. 1980) and the rate of neurotransmitter re-uptake (Pastuszko et al. 1982) are decreased following acute hypoxia. Furthermore, Sulkowski *et al* (2002) observed that ischemia decreases synaptosomal oxygen consumption and metabolic capacity for a period of 24 hours.

Cerebral ischemia induces a marked depletion in synaptic vesicle content and increases the number of damaged mitochondria (Sulkowski et al. 2002, Costain et al. 2008). Importantly, ischemia-induced alterations in synaptosomal morphology are indistinguishable from that of brain slices (Sulkowski et al. 2002). Ischemia-like conditions (hypoxic stress or excitotoxicity) induce dramatic and rapidly reversible structural/morphological changes in dendritic spines (Hasbani et al. 2001, Mattson et al. 1998, Park et al. 1996, Enright et al. 2007,

MacManus 2002), and more recently ischemia induced autophagy has become an active area of interest (Liu et al. 2010). While necrotic cell death is well known to occur in cerebral infarcts, the recent identification of programed necrosis, or 'necroptosis', has reinvigorated

Fig. 1. Outline of the ischemic synaptosomal proteomic analysis procedure. Focal ischemia was performed 20 hours prior to the isolation of the ipsilateral (ischemic) and contralateral (non-ischemic) mouse brain hemispheres. Synaptosomes were isolated from the entire

While cell death mechanisms are typically viewed as pathways involving multiple proteins and organelles, neuronal pathology can also be examined from an organelle centric perspective. Organelle dysfunction can precipitate the initiation of cell death pathways, rather than simply being relay points for signaling events. The essence of this distinction is the difference between extrinsic activation of cell death and internal/intrinsic activation. The primary route of activation of cell death following cerebral ischemia is through intrinsic pathways involving the mitochondria, lysosomes endoplasmic reticulum, and nucleus (Yamashima & Oikawa 2009, Chen et al. 2010, Ankarcrona et al. 1995). Thus, a strategy that involves examining the interaction between organelles during pathological conditions may enable the identification of new targets that can be exploited as therapies for cerebral

forebrain hemispheres and processed as described in the diagram.

ischemia.

research in ischemia-induced necrotic cell death.

Zhang & Murphy 2007). The structural (Park et al. 1996) and biochemical alterations (Martone et al. 1999) at the synapse rapidly return to normal following cessation of ischemic stressors. This initial period of apparent recovery is followed by a period of morphological and biochemical alterations that persist for upwards of 24 hours (Martone et al. 1999). Similarly, dysregulation of synaptic adhesion is observed prior to the onset of neuronal cell death and continues thereafter (Costain et al. 2008). These studies indicate that the synapse is highly responsive to ischemia and is an important modulator of post-ischemic neuronal fate. Signals triggered at the synapse propagate toward the cell body and instigate delayed post-ischemic neuronal death in a process termed as *synaptic apoptosis* (Mattson et al. 1998).

Synaptically localized signals, either anti- or pro-apoptotic, can be propagated to the cell body in both an anterograde and retrograde manner (Mattson & Duan 1999). Apoptotic stimuli have been shown to induce caspase-3 activation, mitochondrial membrane depolarization and phospholipid asymmetry in isolated synaptosomes (Mattson et al. 1998). Similarly, trophic factor withdrawal increases axonal caspase-3 activity, but not within the neuronal soma (Mattson & Duan 1999). In hippocampal neurons, apoptotic signals initiated at the dendrites have been shown to subsequently spread toward the cell body (Mattson & Duan 1999). Synaptic apoptosis may be a mechanism that is necessary for synaptic remodeling under non-pathological conditions as well as contributing to or initiating neuronal apoptosis during pathological conditions. Pro-apoptotic proteins have been found to play a role in non-pathological processes such as neurogenesis, neurite outgrowth and synaptic plasticity (Mattson & Gleichmann 2005). This suggests that signals triggered at the synapse may propagate toward the cell body and instigate postischemic neuronal death.

Cellular protein levels are determined by the balance between the rates of synthesis and degradation, and cerebral ischemia has a pronounced effect on these processes. The transcriptional response to cerebral ischemia has been studied using high throughput methods under a variety of experimental conditions (Gilbert et al. 2003, MacManus et al. 2004). Similarly, cerebral ischemia-induced protein degradation has been examined for a variety of individual proteins. Activation of a variety of proteases, such as members of the caspase, calpain and cathepsin families, is a well-described consequence of cerebral ischemia (Vanderklish & Bahr 2000, Kagedal et al. 2001). Complicating this is the observation that cerebral ischemia causes proteosomal (DeGracia et al. 2002), lysosomal (Costain et al. 2010), mitochondrial (Costain et al. 2010) and endoplasmic reticulum dysfunction (Ge et al. 2007). Thus, it is almost impossible to predict post-ischemic cellular protein levels from gene expression data alone. When focusing on a subcellular structure, such as the synapse, an additional mechanism will impact protein levels. Intracellular transport mechanisms can target a protein to a specific region or be involved in sequestering proteins away from their original location (Zhao et al. 2005, Vanderklish & Bahr 2000). As a result of these factors, the best approach for determining post-ischemic synaptic protein levels is to perform a direct assessment using proteomic methodologies.

An understanding of the cell death processes that are precipitated by exposure to cerebral ischemia is necessary for designing rational therapeutic intervention. Considering that cell death can be mediated by multiple inter-related mechanisms, it is perhaps unsurprising that the majority of single target small molecules have failed in clinical trials for stroke (Ginsberg 2008). The role of apoptotic cell death in cerebral ischemia has long been studied (Hou &

Zhang & Murphy 2007). The structural (Park et al. 1996) and biochemical alterations (Martone et al. 1999) at the synapse rapidly return to normal following cessation of ischemic stressors. This initial period of apparent recovery is followed by a period of morphological and biochemical alterations that persist for upwards of 24 hours (Martone et al. 1999). Similarly, dysregulation of synaptic adhesion is observed prior to the onset of neuronal cell death and continues thereafter (Costain et al. 2008). These studies indicate that the synapse is highly responsive to ischemia and is an important modulator of post-ischemic neuronal fate. Signals triggered at the synapse propagate toward the cell body and instigate delayed post-ischemic neuronal death in a process termed as *synaptic apoptosis* (Mattson et al.

Synaptically localized signals, either anti- or pro-apoptotic, can be propagated to the cell body in both an anterograde and retrograde manner (Mattson & Duan 1999). Apoptotic stimuli have been shown to induce caspase-3 activation, mitochondrial membrane depolarization and phospholipid asymmetry in isolated synaptosomes (Mattson et al. 1998). Similarly, trophic factor withdrawal increases axonal caspase-3 activity, but not within the neuronal soma (Mattson & Duan 1999). In hippocampal neurons, apoptotic signals initiated at the dendrites have been shown to subsequently spread toward the cell body (Mattson & Duan 1999). Synaptic apoptosis may be a mechanism that is necessary for synaptic remodeling under non-pathological conditions as well as contributing to or initiating neuronal apoptosis during pathological conditions. Pro-apoptotic proteins have been found to play a role in non-pathological processes such as neurogenesis, neurite outgrowth and synaptic plasticity (Mattson & Gleichmann 2005). This suggests that signals triggered at the synapse may propagate toward the cell body and instigate post-

Cellular protein levels are determined by the balance between the rates of synthesis and degradation, and cerebral ischemia has a pronounced effect on these processes. The transcriptional response to cerebral ischemia has been studied using high throughput methods under a variety of experimental conditions (Gilbert et al. 2003, MacManus et al. 2004). Similarly, cerebral ischemia-induced protein degradation has been examined for a variety of individual proteins. Activation of a variety of proteases, such as members of the caspase, calpain and cathepsin families, is a well-described consequence of cerebral ischemia (Vanderklish & Bahr 2000, Kagedal et al. 2001). Complicating this is the observation that cerebral ischemia causes proteosomal (DeGracia et al. 2002), lysosomal (Costain et al. 2010), mitochondrial (Costain et al. 2010) and endoplasmic reticulum dysfunction (Ge et al. 2007). Thus, it is almost impossible to predict post-ischemic cellular protein levels from gene expression data alone. When focusing on a subcellular structure, such as the synapse, an additional mechanism will impact protein levels. Intracellular transport mechanisms can target a protein to a specific region or be involved in sequestering proteins away from their original location (Zhao et al. 2005, Vanderklish & Bahr 2000). As a result of these factors, the best approach for determining post-ischemic synaptic protein levels is to perform a direct

An understanding of the cell death processes that are precipitated by exposure to cerebral ischemia is necessary for designing rational therapeutic intervention. Considering that cell death can be mediated by multiple inter-related mechanisms, it is perhaps unsurprising that the majority of single target small molecules have failed in clinical trials for stroke (Ginsberg 2008). The role of apoptotic cell death in cerebral ischemia has long been studied (Hou &

1998).

ischemic neuronal death.

assessment using proteomic methodologies.

MacManus 2002), and more recently ischemia induced autophagy has become an active area of interest (Liu et al. 2010). While necrotic cell death is well known to occur in cerebral infarcts, the recent identification of programed necrosis, or 'necroptosis', has reinvigorated research in ischemia-induced necrotic cell death.

Fig. 1. Outline of the ischemic synaptosomal proteomic analysis procedure. Focal ischemia was performed 20 hours prior to the isolation of the ipsilateral (ischemic) and contralateral (non-ischemic) mouse brain hemispheres. Synaptosomes were isolated from the entire forebrain hemispheres and processed as described in the diagram.

While cell death mechanisms are typically viewed as pathways involving multiple proteins and organelles, neuronal pathology can also be examined from an organelle centric perspective. Organelle dysfunction can precipitate the initiation of cell death pathways, rather than simply being relay points for signaling events. The essence of this distinction is the difference between extrinsic activation of cell death and internal/intrinsic activation. The primary route of activation of cell death following cerebral ischemia is through intrinsic pathways involving the mitochondria, lysosomes endoplasmic reticulum, and nucleus (Yamashima & Oikawa 2009, Chen et al. 2010, Ankarcrona et al. 1995). Thus, a strategy that involves examining the interaction between organelles during pathological conditions may enable the identification of new targets that can be exploited as therapies for cerebral ischemia.

Fig. 2. NanoLC-MS analysis of contralateral (control) and ipsilateral (stroke) samples. Shown are images representing the nanoLC-MS data from each sample, where each spot represents a peptide ion. MatchRx software was used to extract peptide data, align the control and stroke datasets and correct retention time variations. Shown are the merged images before and after MatchRx-dependent correction. More than 5000 ions were detected per sample.

were dissolved in an appropriate volume of denaturing buffer (50 mM Tris-HCl, pH 8.5, 0.1% SDS) to a final protein concentration of 2 mg/mL. One hundred µg of each protein was transferred to a fresh tube. The proteins were reduced using dithiothrietol (4 mM, 10 min at 95 C), alkylated using iodoacetamide (10 mM, 20 min at room temperature in dark), and digested using 5 µg of MS-grade trypsin gold (Promega, 12-18 h at 37 C). The digested peptides were diluted 10-fold in 10 mM KH2PO4, pH 3.0, 25% acetonitrile and loaded onto a cation exchange column (POROS® 50 HS, 50-µm particle size, 4.0 mm x 15 mm, Applied Biosystems) for separation. Five fractions were eluted using step-gradient of 0-350 mM KCl. Each fraction was evaporated to dryness and dissolved in 5% acetronitrile, 1% acetic acid for

analysis by mass spectrometry (MS).

The effects of cerebral ischemia are often, by necessity, described from a highly reductionist point of view, with most studies focusing on specific signaling pathways or individual molecules. Conversely, genomic and proteomic datasets offer the opportunity to expand the scope of understanding and enable the interpretation of systematic responses. While there is a wealth of data available describing neuronal proteins localized in synaptosomes as well as pre-synaptic and post-synaptic preparations, to date studies on the effects of cerebral ischemia on the synaptic proteome are limited (Costain et al. 2008). The aim of this chapter is to integrate new and existing genomic and proteomic datasets to provide a comprehensive understanding of the effect of cerebral ischemia on the function of neuronal organelles, as well as their role in mediating cell death and / or neuroprotection.

### **2. Materials and methods**

### **2.1 Animal care**

A local committee for the Canadian Council on Animal Care approved all procedures using mice. The C57B mice were purchased from Charles River Canada (St-Constant, PQ). Under temporary isoflurane anesthesia, the mice (20-23 g) were subjected to occlusion of the left middle cerebral artery (MCAO) using an intraluminal filament as previously described (Costain et al. 2008). After 1 hr of ischemia, the animals were briefly reanesthetized, the filament withdrawn and wounds sutured. After 20 hrs of reperfusion, mice were briefly anesthetized with isoflurane and the brain rapidly excised and dissected on ice.

### **2.2 Synaptosome preparation**

Contralateral (CT) and ischemic (IS) hemispheres from one mouse were manually homogenized in 2 ml of HM buffer (0.32 M sucrose, 1 mM EDTA, 0.25 mM DTT, 1 U/ml RNasin (Promega)) using a dounce homogenizer, 800 rpm 13 strokes at 4 °C. The homogenates were centrifuged (1000 g for 10 min at 4 C) and the supernatant retained. The pellet was homogenized and centrifuged (as before) a second time. The first and second supernatants were transferred to 2 ml polycarbonate tubes and centrifuged at 20,000 g for 20 min at 4 C. The resultant pellet was resuspended in 2 ml HM buffer using a dounce homogenizer. Discontinuous sucrose:percoll gradients were prepared by layering 2 ml each, in order, of 25% (percoll in HM buffer), 15%, 10% and 3% into 10 ml polycarbonate centrifuge tubes. One ml of the sample was then layered on top of a gradient and centrifuged at 32,000 g for 5 min at 4 C. Five fractions were collected following centrifugation: F1 - 3% percoll (cytoplasm), F2 - interface between 3% and 10% percoll (myelin), F3 - interface between 10% and 15% percoll (small synaptosomes, myelin & mitochondria), F4 - interphase between 15% and 25% percoll (intact synaptosomes), and F5 pellet (mitochondria). Fractions F1 - F4 were made up to 3 ml with HM buffer and centrifuged at 12,000 g for 20 min at 4 C. The supernatant was removed and the pellet washed with 1 x PBS, twice (each time spinning at 12000 x g for 10min). The final pellet was resuspended in either HM buffer or 50 mM Tris pH 8.5, 0.1% SDS. The physical and biochemical characteristics of the synaptosome preparation used here are described in further detail in Costain *et al*. (2008).

### **2.3 Protein preparation, digestion and ion exchange chromatography**

Proteins from each synaptosome sample were precipitated by adding 10 volumes of cold acetone and incubating at 1 h at -20 C followed by centrifugation at 5000g for 5 min. Pellets

The effects of cerebral ischemia are often, by necessity, described from a highly reductionist point of view, with most studies focusing on specific signaling pathways or individual molecules. Conversely, genomic and proteomic datasets offer the opportunity to expand the scope of understanding and enable the interpretation of systematic responses. While there is a wealth of data available describing neuronal proteins localized in synaptosomes as well as pre-synaptic and post-synaptic preparations, to date studies on the effects of cerebral ischemia on the synaptic proteome are limited (Costain et al. 2008). The aim of this chapter is to integrate new and existing genomic and proteomic datasets to provide a comprehensive understanding of the effect of cerebral ischemia on the function of neuronal

A local committee for the Canadian Council on Animal Care approved all procedures using mice. The C57B mice were purchased from Charles River Canada (St-Constant, PQ). Under temporary isoflurane anesthesia, the mice (20-23 g) were subjected to occlusion of the left middle cerebral artery (MCAO) using an intraluminal filament as previously described (Costain et al. 2008). After 1 hr of ischemia, the animals were briefly reanesthetized, the filament withdrawn and wounds sutured. After 20 hrs of reperfusion, mice were briefly