
 pituitary adenylate cyclase activating polypeptide ( pacap ) is a neuropeptide with widespread occurrence in various organs and diverse effects both in the nervous system and in the periphery [ 1 , 2 ] .
pacap is strongly expressed in the central nervous system , where it exerts several effects such that it is a central regulator of circadian rhythmic activities , plays a role in memory formation   and psychiatric processes , and is involved in central feeding control .
pacap is also known to be expressed in the retina , along with its receptors ( pac1 , vpac1 , and vpac2 receptors ) .
numerous studies have provided evidence that the neuroprotective effects are mainly mediated by the pac1 receptor and diverging downstream pathways upon its activation [ 79 ] .
the pac1 receptor has several splice variants , which can mediate not only different but also opposing effects .
several research groups have proven that pacap has strong protective effects against various retinal injuries . in vitro
, it protects retinal explants against excitotoxic injury   and retinal pigment epithelial cells against oxidative stress . in vivo
retina studies show that pacap protects against nmda- and msg - induced excitotoxic damage [ 13 , 14 ] , uv light - induced lesion , and optic nerve lesion .
retinal ischemia can be induced by several methods , mimicking pathological features seen in human glaucoma - related retinal lesions , in chronic retinal hypoperfusion , and in other types of retinal lesions accompanied by ischemia .
the protective role of pacap has also been proven in models of retinal ischemia [ 18 , 19 ] .
based on these studies , evidence for the protective effects of pacap in the retina is now well established . the bioavailability and fast degradation of pacap limit its therapeutic use and attention has been drawn to the application of shorter fragments and/or analogs .
n - terminally shorter fragments of pacap usually have antagonistic effects , but some reports have documented agonistic behavior , depending on the cell / tissue type [ 79 , 21 , 22 ] .
in contrast , c - terminally shorter fragments usually differ in the strength of receptorial binding [ 79 ] .
therefore , it is necessary to test whether shorter pacap fragments have any effect , ameliorating or damaging , on retinal lesions .
we have previously shown that pacap 6 - 38 , the most widely used antagonist of pacap , has an aggravating effect on retinal excitotoxic lesion . however , it is not known , whether the shorter fragments of pacap have any effect on the retina . therefore , the first aim of our present study was to examine the effects of pacap fragments 4 - 13 , 4 - 22 , 6 - 10 , 6 - 15 , 11 - 15 , and 20 - 31 on chronic retinal hypoperfusion induced by bilateral carotid artery occlusion .
possible fragments to be tested were selected in order to cover a wide range of the molecule , from the n - terminal to the c - terminal and middle region of the peptide .
in addition , the n - terminal fragments show a high similarity with the structure of vip .
furthermore , the 4 - 13 domain , for example , shows high selectivity to the pac1 receptor , which is mainly responsible for the neuroprotective effects of pacap [ 79 ] . in a recent study
we have shown that the peptide most closely related to pacap , namely , vasoactive intestinal peptide ( vip ) , is also protective in retinal ischemia . however , to achieve a degree of neuroprotection similar to pacap , higher doses are required .
other members of the peptide family have not been tested in retinal ischemia so far .
therefore , the second aim of the present study was to investigate whether secretin and glucagon have any effect on a rat model of retinal ischemia .
animals were housed in individual cages , fed , and watered ad libitum , under light / dark cycles of 12/12  h. all animal procedures complied with the university of pecs ( number ba02/2000 - 15024/2011 ) for the ethical use of animals .
adult male rats ( n = 32 ) weighing 250300  g were exposed to permanent bilateral common carotid artery occlusion ( bccao ) under isoflurane anesthesia and both common carotid arteries were ligated with a 3 - 0 filament through a midline incision .
a group of animals underwent anesthesia and all steps of the surgical procedure , except ligation of the carotid arteries .
these animals served as sham - operated animals ( n = 7 ) . immediately following the operation , pacap 1 - 38 or its fragments ( pacap 4 - 13 , 4 - 22 , 6 - 10 , 6 - 15 , 11 - 15 , and 20 - 31 ) or other members of the peptide family such as secretin and glucagon ( 100  pmol/5  l ) were injected intravitreally using 30-gauge hamilton syringe into the right vitreous body of animals .
the left eyes received the same volumes of vehicle treatment ( physiological saline ) and served as ischemic eyes .
rats were sacrificed with an overdose of anesthetic after 2 weeks of bccao and the eyes were processed for histological analysis .
briefly , the eyes were dissected in pbs and fixed in 4% paraformaldehyde dissolved in 0.1  m pb . following fixation , tissues were embedded in durcupan acm resin , cut at 2  m , and stained with toluidine blue .
four tissue blocks from at least four animals were prepared and central retinal areas within 1  mm from the optic nerve were used for measurements ( n = 5 measurements from one tissue block ) .
sections where the gcl appeared thicker than a single cell row were omitted from evaluation .
the following parameters were measured : ( i ) the width of the outer and inner nuclear and plexiform layers ( onl , opl , inl , and ipl , resp . ) ; ( ii ) the number of cells/100  m section length in the ganglion cell layer ( gcl ) ; ( iii ) the number of cells/500  m area in the inl .
statistical comparisons were made using the anova test followed by tukey - b 's post hoc analysis .
bccao resulted in severely reduced thickness of retinal layers as observed two weeks after ligation compared to sham - operated controls ( figure 1(a ) ) .
marks of degeneration with individual variations are visible in all retinal layers ( figure 1(b ) ) .
morphometric analysis revealed that the most pronounced reduction in thickness in retinas with bccao was found in the opl ( figure 2 ) .
this layer was significantly thinner than that of the control specimens ( figure 2 ) .
many cells in the ganglion cell layer ( gcl ) also suffered degeneration , shown by necrotic cells in this layer ( figures 1 and 2 ) .
this fact is well reflected in the reduced number of cells in the gcl ( figure 2 ) .
intraocular pacap 1 - 38 treatment following bccao led to nearly healthy appearance of the retinal layers ( figure 1(c ) ) .
the thickness of the major retinal layers was almost identical with that of the sham - operated animals and was significantly larger than that of control ischemic ones ( figure 2 ) .
this was especially conspicuous in the opl , which disappeared , and the onl and inl layers were fused in several control animals and were preserved in all pacap 1 - 38 injected animals .
however , the number of cells in the gcl seemed to be lower than in the sham - operated animals ( figure 2 ) .
intravitreal injection of different pacap fragments ( pacap 4 - 22 , 6 - 15 , 11 - 15 , 20 - 31 , 6 - 10 , and 4 - 13 ) did not ameliorate the ischemic damage of the retina ( figures 1(d)1(i ) ) . in all samples , nuclear layers ( onl , inl ) and plexiform layers ( opl , ipl ) were reduced , and the cell number in the gcl was significantly less than in sham - operated controls . however , injection with pacap 6 - 10 and 4 - 13 resulted in further reduction of the inner plexiform and nuclear layers ( figures 1(h ) and 1(i ) ) .
treatment with members of the pacap - related peptide superfamily , glucagon and secretin , led to no aggravation or amelioration of the ischemic retinal lesion ( figure 3 ) .
pacap is thought to play an important role in visual processing , as it is an important cotransmitter in the retinohypothalamic tract that is the major input for the biological clock situated in the suprachiasmatic nucleus . besides its supposed physiological actions in the retina , pacap is a well - established retinoprotective peptide .
endogenous protection is proven by the increased vulnerability of the retina in mice lacking endogenous pacap in ischemic retinal lesion   and in nmda - induced excitotoxic injury .
these observations are further supported by the increased apoptotic activity in retinas injected by the pacap antagonist pacap 6 - 38 .
as pacap is a well - accepted retinoprotective agent in models of different retinal injuries , several studies have attempted to explore the molecular mechanisms explaining this strong retinoprotection .
we have shown that pacap activates the antiapoptotic , while it inhibits the proapoptotic signaling pathways in retinal toxic lesions . in retinal ischemia
, we have shown that pacap administration provides protection by involving akt , mapk pathways , and anti - inflammatory actions .
given the very potent actions of pacap in retinal and other pathologies , there is an urging demand for developing novel analogues and/or fragments to increase the bioavailability and stability of the peptide for future potential therapeutic use [ 7 , 8 ] .
binding pacap to other carrier molecules is a possibility to increase its capacity to traverse through membranes and to exert cytoprotective effects .
it has also been shown that pacap is able to pass through cell membranes and convey nonreceptor mediated effects .
this has raised the possibility of using pacap fragments also for supporting transport of other nonpenetrable molecules .
several analogues have also been developed for these purposes [ 7 , 8 , 32 ] .
interestingly , however , a stable analogue did not provide stronger neuroprotection in a stroke model than the natural peptide .
unfortunately , all these efforts do not seem to lead to obtaining of a more effective molecule than the original peptide or to obtaining of a biologically more stable analogue that has the same protective property as pacap 1 - 38 .
the evolutionary pressure on the form of pacap 1 - 38 seems to be very strong , as effects found in vertebrates have also been described in invertebrates .
although a lot of pharmacological and receptor binding studies have been performed with fragments / analogues of pacap , only few studies have tested the biological efficacy of these forms . since some studies suggest that there are unconventional binding sites , unusual behavior of the peptide forms in some tissues and even implies the existence of unknown receptor splice variants [ 21 , 22 , 34 ] ; it is important to test the biological effects of fragments in a system that is well standardized in numerous earlier studies .
our present results show that the shorter fragments of pacap , as expected , do not provide any neuroprotection .
in addition , we can confirm that most of them do not deteriorate the degree of damage either , although some parameters were worsened in cases of pacap 6 - 10 and 4 - 13 .
this is in accordance with our earlier observations showing that pacap antagonists pacap 6 - 38 and 6 - 27 lead to aggravated retinal lesion in an msg - induced retinal injury model .
some other members of this family have been localized in the vertebrate retina and several functions have been attributed to them [ 35 , 36 ] .
glucagonergic amacrine cells represent a small subpopulation of the amacrine cells possibly playing a role in the visual processing [ 37 , 38 ] .
other studies have shown that glucagon plays a role in eye bulb growth and glucagon is suggested to be an endogenous mediator of emmetropization .
an early study has shown that glucagon increases camp in chick retinal muller cells , similar to the actions of vip .
the protective effects of secretin and glucagon in cerebral pathologies have been implicated in a few studies [ 4143 ] . in cerebral ischemia , glucagon has been shown to exert neuroprotective effects in vivo .
it has not been investigated so far whether secretin or glucagon is protective in retinal lesions . based on our present study , neither glucagon nor secretin
based on our present and previous results , a strong neuroprotective effect is a specific pacap - action , since vip could exert its protective effects only in much higher doses , while secretin and glucagon did not have any effects .
our present results confirm that the natural form of the peptide , pacap 1 - 38 , is the most effective peptide form in retinal ischemia , and the 38 amino acid form of the peptide can not be replaced by another fragment or another member of the peptide family that we know of .
our results further support the potent retinoprotective effects of pacap and call for further studies to establish the future possible clinical introduction of pacap - related retinoprotective therapeutic approach .
 pituitary adenylate cyclase activating polypeptide ( pacap ) has neuroprotective effects in different neuronal and retinal injuries . 
 retinal ischemia can be effectively modelled by permanent bilateral common carotid artery occlusion ( bccao ) , which causes chronic hypoperfusion - induced degeneration in the entire rat retina . 
 the retinoprotective effect of pacap 1 - 38 and vip is well - established in ischemic retinopathy . 
 however , little is known about the effects of related peptides and pacap fragments in ischemic retinopathy . 
 the aim of the present study was to investigate the potential retinoprotective effects of different pacap fragments ( pacap 4 - 13 , 4 - 22 , 6 - 10 , 6 - 15 , 11 - 15 , and 20 - 31 ) and related peptides ( secretin , glucagon ) in bccao - induced ischemic retinopathy . 
 wistar rats ( 3 - 4 months old ) were used in the experiment . 
 after performing bccao , the right eyes of the animals were treated with pacap fragments or related peptides intravitreal ( 100  pm ) , while the left eyes were injected with saline serving as control eyes . 
 sham - operated ( without bccao ) rats received the same treatment . 
 routine histology was performed 2 weeks after the surgery ; cells were counted and the thickness of retinal layers was compared . 
 our results revealed significant neuroprotection by pacap 1 - 38 but did not reveal retinoprotective effect of the pacap fragments or related peptides . 
 these results suggest that pacap 1 - 38 has the greatest efficacy in ischemic retinopathy . 
1. Introduction
2. Materials and Methods
3. Results and Discussion