In keeping with previous reports, we have found that hippocampal cells express GH under basal conditions and, more interestingly, the number of GH-expressing cells seems to increase after brain injury induced by kainate acid administration in rats (P. Devesa et al., 2011). Furthermore, using a double-labeling immunofluorescence we were able to notice that almost all GH-positive cells also showed BrdU immunoreactivity, thus suggesting the existence of a strong correlation between GH expression and cell proliferation (P. Devesa, 2011) (Figure 3). Interestingly, data from Katakowski et al. (Katakowski et al., 2003) demonstrate that the activation of PI3K/Akt signal transduction pathway mediates the migration of neuroblasts to damaged brain areas after stroke, most likely for inducing regeneration. PI3K/Akt is a key signaling pathway for the intracellular effects of GH (Costoya et al., 1999). Moreover, the group of Scheepens demonstrated that the expression of GH and its receptor is strongly upregulated after brain injury and specifically associated with stressed neurons and glia (Scheepens et al., 2000). More recently, the same group demonstrated that during recovery from an ischemic brain injury, a cerebral growth hormone axis is activated; the level of GHR immunoreactivity in the ipsilateral SVZ was significantly increased 5 days after injury vs. the contralateral SVZ, coinciding both spatially and temporally with injury-induced neurogenesis. The population of GHR immunopositive cells in the ipsilateral SVZ at this time was found to include proliferating cells, neural progenitor cells and post-proliferative

As indicated before, several lines of evidence support a role for GH in neurogenesis. The GH receptor is expressed in regions of the brain in which neurogenesis occurs during embryonic brain development (García-Aragón et al., 1992; Turnley et al., 2002) and in neurogenic

indicate inhibition.

Fig. 2. Factors involved in adult neurogenesis control.

migratory neuroblasts (Christophidis et al., 2009).

administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease (Byts & Sirén, 2009). Brain specific blockade of EPOR leads to deficits in neural cell proliferation and neuronal survival in the embryonic brain and in post-stroke neurogenesis in the adult brain (Chen et al., 2007; Tsai et al., 2006). Moreover, *in vivo* and *in vitro* data indicate hat EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal remyelination and lead to functional recovery after stroke (L. Zhang et al., 2010). Of interest here is that growth hormone induces EPO release from kidneys.

Brain injuries such as ischemia affect adult neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in neurogenic niches. The ischemic insult increases the number of progenitor cells in monkey subgranular and subventricular zones, and causes gliogenesis in the ischemia prone hippocampal CA1 sector (Tonchev, 2011). The analysis of the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors revealed that a fraction of mitotic progenitors were positive for the neurotrophin receptor Tyrosine kinase B (TrkB), while immature neurons expressed the neurotrophin receptor Tyrosine kinase A (TrkA). Astroglia, ependymal cells and blood vessels in subventricular zone were positive for distinctive sets of ligands/receptors indicating that a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate subventricular zone (Tonchev, 2011). The analysis of microglial and astroglial proliferation in postischemic hippocampal CA1 sector showed that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus (Tonchev, 2011). Figure 2 summarizes how these hormones and growth factors act on adult neurogenesis.

#### **2.2.5 Growth hormone/IGF-I system and adult neurogenesis**

GH is a pleiotropic hormone expressed not only in the pituitary but in almost any tissue (Devesa et al., 2010b). Thus, far beyond of its classical actions on body growth and intermediate metabolism, GH exerts an important role in the regulation of cell proliferation and survival in several tissues, including the CNS (Costoya et al., 1999; Sanders et al., 2009; McLenachan et al., 2009; Aberg et al., 2009).

The hypothesis that Growth Hormone (GH) and IGF-I play a role on brain repair after an injury has been postulated years ago. The existence of GH expression within the CNS has been reported by several authors, however its physiological role and, in particular, its possible contribution in the reparation of neurologic injuries remain poorly understood despite of that the positive effects of GH treatment on adult neurogenesis have been demonstrated in laboratory animals (McLenachan et al., 2009; Christophidis et al., 2009; Svensson et al., 2008; P. Devesa et al., 2011), and recent data from our group and others (Devesa et al., 2009; High et al., 2010; Reimunde et al., 2010; Reimunde et al., 2011; Devesa et al., 2011) suggest that the hormone may play a similar role in humans. GH-driven neurogenesis may also depend on the local production of GH (P. Devesa et al., 2011), the so called perypheral GH system that may be activated under both physiological and pathologic conditions (Devesa et al., 2010b).

administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease (Byts & Sirén, 2009). Brain specific blockade of EPOR leads to deficits in neural cell proliferation and neuronal survival in the embryonic brain and in post-stroke neurogenesis in the adult brain (Chen et al., 2007; Tsai et al., 2006). Moreover, *in vivo* and *in vitro* data indicate hat EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal remyelination and lead to functional recovery after stroke (L. Zhang et al., 2010). Of interest here is that growth

Brain injuries such as ischemia affect adult neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in neurogenic niches. The ischemic insult increases the number of progenitor cells in monkey subgranular and subventricular zones, and causes gliogenesis in the ischemia prone hippocampal CA1 sector (Tonchev, 2011). The analysis of the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors revealed that a fraction of mitotic progenitors were positive for the neurotrophin receptor Tyrosine kinase B (TrkB), while immature neurons expressed the neurotrophin receptor Tyrosine kinase A (TrkA). Astroglia, ependymal cells and blood vessels in subventricular zone were positive for distinctive sets of ligands/receptors indicating that a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate subventricular zone (Tonchev, 2011). The analysis of microglial and astroglial proliferation in postischemic hippocampal CA1 sector showed that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus (Tonchev, 2011). Figure 2 summarizes how these hormones and growth factors act on adult neurogenesis.

GH is a pleiotropic hormone expressed not only in the pituitary but in almost any tissue (Devesa et al., 2010b). Thus, far beyond of its classical actions on body growth and intermediate metabolism, GH exerts an important role in the regulation of cell proliferation and survival in several tissues, including the CNS (Costoya et al., 1999; Sanders et al., 2009;

The hypothesis that Growth Hormone (GH) and IGF-I play a role on brain repair after an injury has been postulated years ago. The existence of GH expression within the CNS has been reported by several authors, however its physiological role and, in particular, its possible contribution in the reparation of neurologic injuries remain poorly understood despite of that the positive effects of GH treatment on adult neurogenesis have been demonstrated in laboratory animals (McLenachan et al., 2009; Christophidis et al., 2009; Svensson et al., 2008; P. Devesa et al., 2011), and recent data from our group and others (Devesa et al., 2009; High et al., 2010; Reimunde et al., 2010; Reimunde et al., 2011; Devesa et al., 2011) suggest that the hormone may play a similar role in humans. GH-driven neurogenesis may also depend on the local production of GH (P. Devesa et al., 2011), the so called perypheral GH system that may

be activated under both physiological and pathologic conditions (Devesa et al., 2010b).

hormone induces EPO release from kidneys.

**2.2.5 Growth hormone/IGF-I system and adult neurogenesis**

McLenachan et al., 2009; Aberg et al., 2009).

Blue lines indicate stimulation of neural precursors cells proliferation and/or survival, while red lines indicate inhibition.

Fig. 2. Factors involved in adult neurogenesis control.

In keeping with previous reports, we have found that hippocampal cells express GH under basal conditions and, more interestingly, the number of GH-expressing cells seems to increase after brain injury induced by kainate acid administration in rats (P. Devesa et al., 2011). Furthermore, using a double-labeling immunofluorescence we were able to notice that almost all GH-positive cells also showed BrdU immunoreactivity, thus suggesting the existence of a strong correlation between GH expression and cell proliferation (P. Devesa, 2011) (Figure 3). Interestingly, data from Katakowski et al. (Katakowski et al., 2003) demonstrate that the activation of PI3K/Akt signal transduction pathway mediates the migration of neuroblasts

to damaged brain areas after stroke, most likely for inducing regeneration. PI3K/Akt is a key signaling pathway for the intracellular effects of GH (Costoya et al., 1999). Moreover, the group of Scheepens demonstrated that the expression of GH and its receptor is strongly upregulated after brain injury and specifically associated with stressed neurons and glia (Scheepens et al., 2000). More recently, the same group demonstrated that during recovery from an ischemic brain injury, a cerebral growth hormone axis is activated; the level of GHR immunoreactivity in the ipsilateral SVZ was significantly increased 5 days after injury vs. the contralateral SVZ, coinciding both spatially and temporally with injury-induced neurogenesis. The population of GHR immunopositive cells in the ipsilateral SVZ at this time was found to include proliferating cells, neural progenitor cells and post-proliferative migratory neuroblasts (Christophidis et al., 2009).

As indicated before, several lines of evidence support a role for GH in neurogenesis. The GH receptor is expressed in regions of the brain in which neurogenesis occurs during embryonic brain development (García-Aragón et al., 1992; Turnley et al., 2002) and in neurogenic

Growth Hormone and Kynesitherapy for Brain Injury Recovery 429

Neurospheres derived from NSCs obtained from the dentate gyrus of 9days old mice in proliferation medium showing irGH (left, green) and irGHR (right, green dots). The detection of ir for Nestin (red) indicates that cells in neurospheres are undifferentiated or in development. Taken from Pablo Devesa,

Neurospheres derived from NSCs obtained from the dentate gyrus of 9days old mice in proliferation medium showing irGH (left, green) and irGHR (right, green dots). The detection of ir for SOX2 (red) indicates that cells in neurospheres are undifferentiated or in development. Taken from Pablo Devesa,

Fig. 5. Confocal microscopy. Colocalization of GH and GHR with SOX2 in mice

Fig. 4. Confocal microscopy. Colocalization of GH and GHR with Nestin in mice

Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

neurospheres.

neurospheres.

regions of the postnatal rat brain (Lobie et al., 1993). Growth hormone itself is also found in cells of the ventricular zone during embryonic neurogenesis (Turnley et al., 2002), and is produced endogenously within the postnatal hippocampus (Donahue et al., 2002; Donahue et al., 2006; Sun et al., 2005a; Sun et al., 2005b). Interestingly, GH gene expression within the hippocampus is increased by some factors known to increase neurogenesis (Parent, 2003), including learning (Donahue et al., 2002) and estrogen (Donahue et al., 2006).

The detection of irBrDU in the nucleus of the cell (green) indicated by the arrow demonstrates that it is a newly born cell in rat hippocampal CA3 area showing irGH (brown), after brain injury induced by kainate acid administration and GH treatment, in its cytoplasm. Newly born neurons formed in the subgranular layer of the dentate gyrus migrate to the CA3 zone after commencing to maturate in the granular area. Thus, it is feasible that the detection of irGH in CA3 cells may be related to a trophic and survival role of the hormone. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 3. Immunofluorescence (60x). Colocalization of GH and BrdU in a newly born hippocampal cell.

However, the role of GH in the hippocampal dentate gyrus has been related to neuronal survival rather than generation, as altered hippocampal GH levels have no effect on cell proliferation but do affect the survival of immature neurons (Sun et al., 2005b; Sun et al., 2007; Lichtenwalner et al., 2006). Studies of the effects of GH on embryonic rat cerebral cortical (Ajo et al., 2003) and hippocampal (Byts et al., 2008) neuronal cultures found that it induces the proliferation and differentiation of these cells. Overall, these findings indicate that GH may facilitate the proliferation, differentiation and survival of new neurons in response to brain injury. Further studies will reveal whether or not GH is "called" for brain repair after GPR17 activation.

On these bases, we explored a potential role for GH and its receptor on the proliferation, differentiation and survival of neural stem cells (NSCs) *in vitro*. NSCs were isolated from the SGZ of the DG from 9 days old C57/BL6 mice and cultured as neurospheres.

Western blot and immunofluorescence studies showed that neurospheres derived from these NSCs were demonstrated to express both GH and its receptor either in conditions of proliferation and differentiation showing colocalization with nestin (Figure 4) and SOX2 (Figure 5), markers of undifferentiated cells or cells in development. Migrating cells from neurospheres also showed irGHR (Figure 6). In all, these results indicate that GH and its receptor seem to be needed for the physiological neurogenesis.

regions of the postnatal rat brain (Lobie et al., 1993). Growth hormone itself is also found in cells of the ventricular zone during embryonic neurogenesis (Turnley et al., 2002), and is produced endogenously within the postnatal hippocampus (Donahue et al., 2002; Donahue et al., 2006; Sun et al., 2005a; Sun et al., 2005b). Interestingly, GH gene expression within the hippocampus is increased by some factors known to increase neurogenesis (Parent, 2003),

The detection of irBrDU in the nucleus of the cell (green) indicated by the arrow demonstrates that it is a newly born cell in rat hippocampal CA3 area showing irGH (brown), after brain injury induced by kainate acid administration and GH treatment, in its cytoplasm. Newly born neurons formed in the subgranular layer of the dentate gyrus migrate to the CA3 zone after commencing to maturate in the granular area. Thus, it is feasible that the detection of irGH in CA3 cells may be related to a trophic and survival role of the hormone. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de

However, the role of GH in the hippocampal dentate gyrus has been related to neuronal survival rather than generation, as altered hippocampal GH levels have no effect on cell proliferation but do affect the survival of immature neurons (Sun et al., 2005b; Sun et al., 2007; Lichtenwalner et al., 2006). Studies of the effects of GH on embryonic rat cerebral cortical (Ajo et al., 2003) and hippocampal (Byts et al., 2008) neuronal cultures found that it induces the proliferation and differentiation of these cells. Overall, these findings indicate that GH may facilitate the proliferation, differentiation and survival of new neurons in response to brain injury. Further studies will reveal whether or not GH is "called" for brain

On these bases, we explored a potential role for GH and its receptor on the proliferation, differentiation and survival of neural stem cells (NSCs) *in vitro*. NSCs were isolated from the

Western blot and immunofluorescence studies showed that neurospheres derived from these NSCs were demonstrated to express both GH and its receptor either in conditions of proliferation and differentiation showing colocalization with nestin (Figure 4) and SOX2 (Figure 5), markers of undifferentiated cells or cells in development. Migrating cells from neurospheres also showed irGHR (Figure 6). In all, these results indicate that GH and its

SGZ of the DG from 9 days old C57/BL6 mice and cultured as neurospheres.

receptor seem to be needed for the physiological neurogenesis.

Fig. 3. Immunofluorescence (60x). Colocalization of GH and BrdU in a newly born

Compostela, Spain.

hippocampal cell.

repair after GPR17 activation.

including learning (Donahue et al., 2002) and estrogen (Donahue et al., 2006).

Neurospheres derived from NSCs obtained from the dentate gyrus of 9days old mice in proliferation medium showing irGH (left, green) and irGHR (right, green dots). The detection of ir for Nestin (red) indicates that cells in neurospheres are undifferentiated or in development. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 4. Confocal microscopy. Colocalization of GH and GHR with Nestin in mice neurospheres.

Neurospheres derived from NSCs obtained from the dentate gyrus of 9days old mice in proliferation medium showing irGH (left, green) and irGHR (right, green dots). The detection of ir for SOX2 (red) indicates that cells in neurospheres are undifferentiated or in development. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 5. Confocal microscopy. Colocalization of GH and GHR with SOX2 in mice neurospheres.

Growth Hormone and Kynesitherapy for Brain Injury Recovery 431

accelerated neuronal differentiation (McLenachan et al., 2008). Interestingly, treatment of neurospheres derived from newborn or adult mouse neural stem cells with 100 ng/mL rat GH, which stimulates proliferation (Christophidis et al., 2009), significantly reduced

GH treatment increased the number of proliferating stem cells in proliferation conditions, as irBrdU (red) shows. Left: control, saline treated cells. Right: GH treated cells. Taken from Pablo Devesa,

Overall, it appears that the effect of GH on neuronal differentiation depends upon the

We also analyzed the role of GH on NSCs survival. In basal conditions there is a physiological rate of apoptosis that is significantly reduced by adding GH to the culture media in differentiation conditions. The antiapoptotic role of the hormone was clearly demonstrated by treating the cells with the GH agonist pegvisomant. This drug binds to the GHR but is unable to induce any kind of intracellular response because inhibits the dimerization of the GHR needed for intracellular signalling. In these conditions, basal apoptosis was significantly increased indicating that the autocrine interplay GH—GHR is key for NSCs survival. Moreover, treating the cells with GH did not modify the increased

GH effects are mediated via the GHR, which is a member of the cytokine receptor superfamily. The critical step in initiating GH signaling is the activation of receptorassociated Janus kinase 2 (JAK2), which induces cross-phosphorylation of tyrosine residues in the kinase domain of JAK2 and GHR. Phosphorylated residues on JAK2 and GHR form docking sites for the members of the STAT family of transcription factors (Perrini et al., 2008). Phosphorylation of the STATs by JAK2 results in their dissociation from the receptor and translocation to the nucleus, with subsequent binding to DNA and regulation of gene expression. Among the target genes, GH regulates the expression of suppressor of cytokine signaling (SOCS), a family of negative regulators that terminate the GH signaling cascade (Hansen et al., 1999). SOCS proteins bind to phosphotyrosine residues on the GHR or JAK2

neuronal differentiation (Turnley et al., 2002; Scott et al., 2006).

Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

apoptosis elicited by pegvisomant (Figure 8).

concentration of GH and proliferative potential of the cells used.

Fig. 7. *In vitro* effects of GH treatment on proliferating neural stem cells.

Detection of irGHR (red dots) in different cells migrating from neurospheres formed from NSCs obtained from the dentate gyrus of 9days old mice. The detection of irGFAP (left, green) indicates that these cells are astrocytes, while the detection of irPSANCAM (right, green) indicates that this cell is a migrating neuron. Cells nuclei are stained in blue. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 6. Confocal microscopy. Detection of GHR in cells migrating from mice neurospheres.

However, from these studies we could not be able for establishing the exact role that the GH—GHR system plays on this mechanism. This is why we explored what would happen when adding the hormone to the culture media either in conditions of proliferation and differentiation.

As reported in other studies (Christophidis et al., 2009) treating NSCs with GH in the presence of BrdU significantly increased the proportion of cells incorporating BrdU almost doubling it (Figure 7). That is, despite of the fact that NSCs express the hormone, the addition of exogenous GH increased stem cells proliferation. This effect is similar to that we observed in animals with kainate acid-induced brain injury (P. Devesa et al., 2011).

It has been suggested that the GHR may actually play a greater role in the survival, migration or differentiation of neurons generated in response to hypoxia/ischemia than in proliferation (Christophidis et al., 2009). Consistent with a role for GHR in survival, GH protects both mature neurons (Möderscheim et al., 2007; Byts et al., 2008; Silva et al., 2003) and primary neurospheres derived from embryonic mouse NSCs (van Marle et al., 2005) from death *in vitro*. Also, the survival of newborn neurons in the subgranular zone of adult rat dentate gyrus is impaired as a result of GH deficiency (Lichtenwalner et al., 2006), and elevated GH levels within the hippocampus reduce apoptosis (Sun et al., 2007). A role for GH in neuronal differentiation is supported by a recent study that found that physiological concentrations of human GH stimulate neurite initiation and arborization of embryonic hippocampal neurons by activating the PI3K/Akt signalling pathway (Byts et al., 2008). Other studies have found that high concentrations of human GH reduce neuronal proliferation but enhance differentiation of these cells instead (Ajo et al., 2003; Lyuh et al., 2007). Indeed, it appears that GH promotes proliferation of neural cells at the expense of their differentiation, as neurosphere cultures derived from GHR *knockout* mice proliferate less than those of *wt* mice due to their inability to respond to autocrine GH, yet they exhibit

Detection of irGHR (red dots) in different cells migrating from neurospheres formed from NSCs obtained from the dentate gyrus of 9days old mice. The detection of irGFAP (left, green) indicates that these cells are astrocytes, while the detection of irPSANCAM (right, green) indicates that this cell is a migrating neuron. Cells nuclei are stained in blue. Taken from Pablo Devesa, Doctoral thesis, 2011.

Fig. 6. Confocal microscopy. Detection of GHR in cells migrating from mice neurospheres. However, from these studies we could not be able for establishing the exact role that the GH—GHR system plays on this mechanism. This is why we explored what would happen when adding the hormone to the culture media either in conditions of proliferation and

As reported in other studies (Christophidis et al., 2009) treating NSCs with GH in the presence of BrdU significantly increased the proportion of cells incorporating BrdU almost doubling it (Figure 7). That is, despite of the fact that NSCs express the hormone, the addition of exogenous GH increased stem cells proliferation. This effect is similar to that we

It has been suggested that the GHR may actually play a greater role in the survival, migration or differentiation of neurons generated in response to hypoxia/ischemia than in proliferation (Christophidis et al., 2009). Consistent with a role for GHR in survival, GH protects both mature neurons (Möderscheim et al., 2007; Byts et al., 2008; Silva et al., 2003) and primary neurospheres derived from embryonic mouse NSCs (van Marle et al., 2005) from death *in vitro*. Also, the survival of newborn neurons in the subgranular zone of adult rat dentate gyrus is impaired as a result of GH deficiency (Lichtenwalner et al., 2006), and elevated GH levels within the hippocampus reduce apoptosis (Sun et al., 2007). A role for GH in neuronal differentiation is supported by a recent study that found that physiological concentrations of human GH stimulate neurite initiation and arborization of embryonic hippocampal neurons by activating the PI3K/Akt signalling pathway (Byts et al., 2008). Other studies have found that high concentrations of human GH reduce neuronal proliferation but enhance differentiation of these cells instead (Ajo et al., 2003; Lyuh et al., 2007). Indeed, it appears that GH promotes proliferation of neural cells at the expense of their differentiation, as neurosphere cultures derived from GHR *knockout* mice proliferate less than those of *wt* mice due to their inability to respond to autocrine GH, yet they exhibit

observed in animals with kainate acid-induced brain injury (P. Devesa et al., 2011).

University of Santiago de Compostela, Spain.

differentiation.

accelerated neuronal differentiation (McLenachan et al., 2008). Interestingly, treatment of neurospheres derived from newborn or adult mouse neural stem cells with 100 ng/mL rat GH, which stimulates proliferation (Christophidis et al., 2009), significantly reduced neuronal differentiation (Turnley et al., 2002; Scott et al., 2006).

GH treatment increased the number of proliferating stem cells in proliferation conditions, as irBrdU (red) shows. Left: control, saline treated cells. Right: GH treated cells. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 7. *In vitro* effects of GH treatment on proliferating neural stem cells.

Overall, it appears that the effect of GH on neuronal differentiation depends upon the concentration of GH and proliferative potential of the cells used.

We also analyzed the role of GH on NSCs survival. In basal conditions there is a physiological rate of apoptosis that is significantly reduced by adding GH to the culture media in differentiation conditions. The antiapoptotic role of the hormone was clearly demonstrated by treating the cells with the GH agonist pegvisomant. This drug binds to the GHR but is unable to induce any kind of intracellular response because inhibits the dimerization of the GHR needed for intracellular signalling. In these conditions, basal apoptosis was significantly increased indicating that the autocrine interplay GH—GHR is key for NSCs survival. Moreover, treating the cells with GH did not modify the increased apoptosis elicited by pegvisomant (Figure 8).

GH effects are mediated via the GHR, which is a member of the cytokine receptor superfamily. The critical step in initiating GH signaling is the activation of receptorassociated Janus kinase 2 (JAK2), which induces cross-phosphorylation of tyrosine residues in the kinase domain of JAK2 and GHR. Phosphorylated residues on JAK2 and GHR form docking sites for the members of the STAT family of transcription factors (Perrini et al., 2008). Phosphorylation of the STATs by JAK2 results in their dissociation from the receptor and translocation to the nucleus, with subsequent binding to DNA and regulation of gene expression. Among the target genes, GH regulates the expression of suppressor of cytokine signaling (SOCS), a family of negative regulators that terminate the GH signaling cascade (Hansen et al., 1999). SOCS proteins bind to phosphotyrosine residues on the GHR or JAK2

Growth Hormone and Kynesitherapy for Brain Injury Recovery 433

1999). Inhibiting PI3K/Akt with a potent inibitor of phosphoinositide 3-kinases led to a significant increase in apoptosis in NSCs, however GH treatment was able to clearly decrease the rate of apoptosis observed by blocking PI3K/Akt signaling. This indicates that the hormone is able for signaling through other different pathways acting on cell survival (P. Devesa, 2011). We then studied the effects of inhibiting another cell survival pathway such as ERK. Inhibiting ERK phosphorylation significantly increased NSCs apoptosis in differentiation conditions, but again the treatment with GH was able to revert the

Since GH was able to revert the propapoptotic effects of the inhibition of both PI3K/Akt and ERK, we decided to study signalling pathways located upstream, specifically mTOR. This is a master regulator of cell mass and metabolism, which is in part regulated by growth factor signalling through the canonical RTK (receptor tyrosine kinase)-PI3K/Akt axis and by

Rapamycin is an allosteric mTOR inhibitor. As expected, treating NSCs with rapamycin led to a significant increase in apoptosis, but once more this effect on cellular death was reverted when GH was added together with rapamycin. This seems to be surprising given the importance of mTOR in signalling cell survival, and again indicated that the hormone uses different pathways for promoting cell survival. However, evidence exists showing that mTOR inhibition can lead to pathway reactivation: abrogation of the negative-feedback loop which is normally initiated by the direct substrate p70S6K (p70 S6 kinase) on insulin receptor substrate proteins can lead to strong PI3K/Akt pathway reactivation, most likely producing ERK pathway reactivation in a PI3K/Akt dependent manner (Carracedo et al., 2008). For a better understanding of such a concept see the scheme shown in Figure 9. Thus, while PI3K/Akt inhibition would allow GH to act through ERK pathway, ERK inhibition would not impede PI3K/Akt to be the survival-signalling pathway. Of interest here is the fact that both kind of inhibitions only were reverted when exogenous GH was added. This means that the autocrine production of the hormone by NSCs is not enough for

We then blocked simultaneously ERK and mTOR activation. Once more the intense apoptosis observed after pharmachological blockade of these pathways was reverted when GH was added together with them. The only explanations for this result is that the hormone may act directly at a upper level in the stream of signaling pathways for cell survival or the effect is due to the overactivation of PI3K occurred as a result of blocking those two other signaling pathways. To test the first possibility we used SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase (Bennett et al., 2001). That is we were acting at the terminal level in the stream. In this case the clear increase in apoptosis produced by the inhibitor could not be reverted by administering GH together with the drug. It suggests either a direct effect of the hormone on JNK activation for cell survival or a direct effect of

In summary, our results (P. Devesa, 2011), together with other studies previously described, demonstrate that GH is able for promoting NSCs proliferation, and also plays a key role on the survival of these cells. With regard to the antiapoptotic role of GH, it seems to be exerted trough different signaling pathways: PI3K/Akt, ERK and pJNK (Figure 10). Since culture media contain insulin our results do not allow us to conclude whether the effect of GH on pJNK is a direct effect or it results from the known crosstalk between GH and insulin at the cellular level (Xu & Messina, 2009) or from the overactivation of PI3K directly enhancing JNK activity (Zhu et al., 1998). In any case GH effects a very strong effect on the survival of NSCs.

proapoptotic effect of blocking ERK signaling.

nutrient (through class III PI3Ks), hypoxia or AMP.

blocking intense cell death signals.

overactivated PI3K on JNK (as postulated by Zhu et al., 1998).

and suppress GH signaling by inhibiting JAK2 activity, competing with STATs for binding to the GHR, or inducing degradation of the GHR complex.

GH administration significantly reduced basal apoptosis in cultured NSCs cells (\*p < 0.01 vs. control). Treatment with pegvisomant abolished the antiapoptotic effect of GH. C: control; P: pegvisomant. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 8. *In vitro* effects of GH treatment on the survival of mice neural stem cells.

The RAS/mitogen-activated protein kinase (MAPK) pathway has also been shown to be activated by GH. The GHR–JAK2 complex has been shown to recruit the adapter protein SHC, resulting in SHC tyrosine phosphorylation and binding to GRB2, and activation of RAS, RAF, MAPK/extracellular-regulated protein kinase (MEK), and ERK-1/2 (Vanderkuur et al., 1997). Alternatively, it has been suggested that GH might also regulate the activation of ERK by a SRC-dependent, JAK2-independent mechanism which involves phopholipase D (Zhu et al., 2002). This JAK2- independent mechanism has been recently demonstrated *in vivo* (Barclay et al., 2010). Tyrosine phosphorylation of a GRB2-binding site in the epidermal growth factor receptor could also be involved in GH-mediated MAPK activation (Yamauchi et al., 1997).

GH signaling via SHC and ERK can be interrupted after blocking insulin receptor substrate-1 (IRS-1) (Wang et al., 2009). IRS-1 is a docking protein tyrosine phosphorylated in response to insulin, IGF-I, GH, and other cytokines. IRS-1 greatly enhances GH-induced ERK.

GH has also been shown to stimulate the PI3K pathway through JAK2-mediated tyrosine phosphorylation of the insulin receptor substrates (IRS-1 to IRS-3), leading to their association with PI3K regulatory subunits (Zhu et al., 2001). In addition, direct binding of the p85 and p85β subunits to phosphotyrosine residues in the carboxyl terminus domain of the GHR has also been demonstrated (Moutoussamy et al., 1998).

As stated before, GH stimulation of PI3K is linked to the stimulation of the antiapoptotic serine protein kinase B or Akt (Costoya et al., 1999). Akt activation has been shown to be dependent on the presence of the JAK2-binding region of GHR, and to promote cell survival through the inhibition of the proapoptotic protein caspase 3 (Sanders et al., 2006). The role of PI3K/Akt on cell survival is well known after the pioneer study of our group (Costoya et al.,

and suppress GH signaling by inhibiting JAK2 activity, competing with STATs for binding

GH administration significantly reduced basal apoptosis in cultured NSCs cells (\*p < 0.01 vs. control). Treatment with pegvisomant abolished the antiapoptotic effect of GH. C: control; P: pegvisomant. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

The RAS/mitogen-activated protein kinase (MAPK) pathway has also been shown to be activated by GH. The GHR–JAK2 complex has been shown to recruit the adapter protein SHC, resulting in SHC tyrosine phosphorylation and binding to GRB2, and activation of RAS, RAF, MAPK/extracellular-regulated protein kinase (MEK), and ERK-1/2 (Vanderkuur et al., 1997). Alternatively, it has been suggested that GH might also regulate the activation of ERK by a SRC-dependent, JAK2-independent mechanism which involves phopholipase D (Zhu et al., 2002). This JAK2- independent mechanism has been recently demonstrated *in vivo* (Barclay et al., 2010). Tyrosine phosphorylation of a GRB2-binding site in the epidermal growth factor receptor could also be involved in GH-mediated MAPK activation (Yamauchi

GH signaling via SHC and ERK can be interrupted after blocking insulin receptor substrate-1 (IRS-1) (Wang et al., 2009). IRS-1 is a docking protein tyrosine phosphorylated in response

GH has also been shown to stimulate the PI3K pathway through JAK2-mediated tyrosine phosphorylation of the insulin receptor substrates (IRS-1 to IRS-3), leading to their association with PI3K regulatory subunits (Zhu et al., 2001). In addition, direct binding of the p85 and p85β subunits to phosphotyrosine residues in the carboxyl terminus domain of

As stated before, GH stimulation of PI3K is linked to the stimulation of the antiapoptotic serine protein kinase B or Akt (Costoya et al., 1999). Akt activation has been shown to be dependent on the presence of the JAK2-binding region of GHR, and to promote cell survival through the inhibition of the proapoptotic protein caspase 3 (Sanders et al., 2006). The role of PI3K/Akt on cell survival is well known after the pioneer study of our group (Costoya et al.,

to insulin, IGF-I, GH, and other cytokines. IRS-1 greatly enhances GH-induced ERK.

the GHR has also been demonstrated (Moutoussamy et al., 1998).

Fig. 8. *In vitro* effects of GH treatment on the survival of mice neural stem cells.

et al., 1997).

to the GHR, or inducing degradation of the GHR complex.

1999). Inhibiting PI3K/Akt with a potent inibitor of phosphoinositide 3-kinases led to a significant increase in apoptosis in NSCs, however GH treatment was able to clearly decrease the rate of apoptosis observed by blocking PI3K/Akt signaling. This indicates that the hormone is able for signaling through other different pathways acting on cell survival (P. Devesa, 2011). We then studied the effects of inhibiting another cell survival pathway such as ERK. Inhibiting ERK phosphorylation significantly increased NSCs apoptosis in differentiation conditions, but again the treatment with GH was able to revert the proapoptotic effect of blocking ERK signaling.

Since GH was able to revert the propapoptotic effects of the inhibition of both PI3K/Akt and ERK, we decided to study signalling pathways located upstream, specifically mTOR. This is a master regulator of cell mass and metabolism, which is in part regulated by growth factor signalling through the canonical RTK (receptor tyrosine kinase)-PI3K/Akt axis and by nutrient (through class III PI3Ks), hypoxia or AMP.

Rapamycin is an allosteric mTOR inhibitor. As expected, treating NSCs with rapamycin led to a significant increase in apoptosis, but once more this effect on cellular death was reverted when GH was added together with rapamycin. This seems to be surprising given the importance of mTOR in signalling cell survival, and again indicated that the hormone uses different pathways for promoting cell survival. However, evidence exists showing that mTOR inhibition can lead to pathway reactivation: abrogation of the negative-feedback loop which is normally initiated by the direct substrate p70S6K (p70 S6 kinase) on insulin receptor substrate proteins can lead to strong PI3K/Akt pathway reactivation, most likely producing ERK pathway reactivation in a PI3K/Akt dependent manner (Carracedo et al., 2008). For a better understanding of such a concept see the scheme shown in Figure 9.

Thus, while PI3K/Akt inhibition would allow GH to act through ERK pathway, ERK inhibition would not impede PI3K/Akt to be the survival-signalling pathway. Of interest here is the fact that both kind of inhibitions only were reverted when exogenous GH was added. This means that the autocrine production of the hormone by NSCs is not enough for blocking intense cell death signals.

We then blocked simultaneously ERK and mTOR activation. Once more the intense apoptosis observed after pharmachological blockade of these pathways was reverted when GH was added together with them. The only explanations for this result is that the hormone may act directly at a upper level in the stream of signaling pathways for cell survival or the effect is due to the overactivation of PI3K occurred as a result of blocking those two other signaling pathways. To test the first possibility we used SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase (Bennett et al., 2001). That is we were acting at the terminal level in the stream. In this case the clear increase in apoptosis produced by the inhibitor could not be reverted by administering GH together with the drug. It suggests either a direct effect of the hormone on JNK activation for cell survival or a direct effect of overactivated PI3K on JNK (as postulated by Zhu et al., 1998).

In summary, our results (P. Devesa, 2011), together with other studies previously described, demonstrate that GH is able for promoting NSCs proliferation, and also plays a key role on the survival of these cells. With regard to the antiapoptotic role of GH, it seems to be exerted trough different signaling pathways: PI3K/Akt, ERK and pJNK (Figure 10). Since culture media contain insulin our results do not allow us to conclude whether the effect of GH on pJNK is a direct effect or it results from the known crosstalk between GH and insulin at the cellular level (Xu & Messina, 2009) or from the overactivation of PI3K directly enhancing JNK activity (Zhu et al., 1998). In any case GH effects a very strong effect on the survival of NSCs.

Growth Hormone and Kynesitherapy for Brain Injury Recovery 435

A number of studies indicate that the neurobiological consequences of the decline in GH/IGF-I, that occurs physiologically during aging, include decreased neurogenesis in the dentate gyrus of the hippocampus (Lichtenwalner et al., 2001; Lichtenwalner et al., 2006), where IGF-I appears to affect primarily the survival of newborn neurons, but also may influence the maturation and differentiation of newborn cells (Aberg et al., 2000;

Despite that neurogenesis is dramatically reduced during senescence, newborn granule cells in the aged dentate gyrus retain the capacity for participation in functional hippocampal networks (Marrone et al., 2011); this is in agreement with the aforementioned hypothesis, suggesting that it is the age-associated lack of neurotrophic factors (GH/IGF-I?) the cause of decreased neurogenesis. In fact, growth hormone prevents neuronal loss in the aged rat

GH induces IGF-I expression in liver and other tissues; however it is not the only factor responsible for it. In fact, there are a number of pathological situations in which a clear divergence exists between GH production and plasma levels of IGF-I. This is the case, for instance, of Anorexia Nervosa; in these patients, while large burst of pituitary GH are released into the blood, IGF-I plasma levels are consistently lower than normal. The opposite situation can be observed in obese children; GH secretion is deficient in these children, but plasma IGF-I levels are normal and growth velocity is above the mean for age (Devesa et al., 1992). The common link between these two situations seems to be plasma glucose levels, low in Anorexia Nervosa and showing a tendency to hyperglycemia in obesity. Thus, it has been postulated that glucose is needed for the hepatic production of IGF-I; more particularly a product of intracellular metabolism of glucose, since 2 deoxiglucose (who can not be metabolized) is unable to induce hepatic IGF-I expression. Therefore, although GH usually is the factor responsible for the hepatic production of IGF-I, in terms of neural effects both hormones may follow different paths. In fact, studies in rats submitted to moderate and severe hypoxia showed that the spatial distribution of the neuroprotection conveyed by growth hormone correlates with the spatial distribution of the constitutive neural growth hormone receptor, but not with the neuroprotection offered by IGF-I treatment in this model. These results suggest that some of the neuroprotective effects of growth hormone are mediated directly through the growth hormone receptor and do not

The effects of the GH/IGF-I axis on cell turnover in the adult brain probably are not limited to neuronal progenitors, since IGF-I can promote proliferation of oligodendrocyte progenitor cells (OPCs) and differentiation and survival of oligodendrocytes (McMorris & McKinnon, 1996; Mason et al., 2000; Aberg et al., 2007; Pang et al., 2007), an effect also induced by EGF (Gonzalez-Perez & Alvarez-Buylla, 2011) whose expression and that of its receptor may also be induced by GH (Pan et al., 2011). Thus, it is likely that the agingrelated decline in GH/IGF-I and dependent changes in oligodendrocyte genesis and/or maturation may contribute to impaired remyelination in the central nervous system of aged individuals (Gilson & Blakemore, 1993; Shields et al., 1999; Franklin et al., 2002; Sim et al., 2002) and to a decline in normal cognitive function. In fact, GH has positive cognitive effects

In all, the system GH/IGF-I exerts both neuroprotective and regenerative effects at the CNS. In line with this a recent study in human patients describes that a high serum IGF-I during the rehabilitation phase of stroke correlates to better recovery of long-term function (Aberg

Darnaudery et al., 2006).

hippocampus (Azcoitia et al., 2005).

involve IGF-I induction (Scheepens et al., 2001).

when given to GH-deficient patients.

et al., 2011).

As showed in the scheme, rapamycin treatment inhibits mTOR, thus leading to a feedback activation of the PI3K/Akt pathway [2]. Physiologically this would lead to increased ERK activation [3], but this not occurs unless exogenous GH is given together with rapamycin [1]. Red lines indicate inhibition; blue lines indicate activation. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 9. Schematic representation of the cell survival signaling pathways studied.

The scheme shows how GH may act on cell survival in NSCs according to the results we obtained. The possibility of a direct effect of the hormone on JNK phosphorylation could not be demonstrated here; another possibility is that the activation of JNK occurs because of overactivation of PI3K after mTOR and ERK blockade. Red line indicates inhibition; blue lines indicate activation. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 10. Schematic representation about the putative role of GH in NSCs survival.

As showed in the scheme, rapamycin treatment inhibits mTOR, thus leading to a feedback activation of the PI3K/Akt pathway [2]. Physiologically this would lead to increased ERK activation [3], but this not occurs unless exogenous GH is given together with rapamycin [1]. Red lines indicate inhibition; blue lines indicate activation. Taken from Pablo Devesa, Doctoral thesis, 2011. University of Santiago de

The scheme shows how GH may act on cell survival in NSCs according to the results we obtained. The possibility of a direct effect of the hormone on JNK phosphorylation could not be demonstrated here; another possibility is that the activation of JNK occurs because of overactivation of PI3K after mTOR and ERK blockade. Red line indicates inhibition; blue lines indicate activation. Taken from Pablo

Devesa, Doctoral thesis, 2011. University of Santiago de Compostela, Spain.

Fig. 10. Schematic representation about the putative role of GH in NSCs survival.

Fig. 9. Schematic representation of the cell survival signaling pathways studied.

Compostela, Spain.

A number of studies indicate that the neurobiological consequences of the decline in GH/IGF-I, that occurs physiologically during aging, include decreased neurogenesis in the dentate gyrus of the hippocampus (Lichtenwalner et al., 2001; Lichtenwalner et al., 2006), where IGF-I appears to affect primarily the survival of newborn neurons, but also may influence the maturation and differentiation of newborn cells (Aberg et al., 2000; Darnaudery et al., 2006).

Despite that neurogenesis is dramatically reduced during senescence, newborn granule cells in the aged dentate gyrus retain the capacity for participation in functional hippocampal networks (Marrone et al., 2011); this is in agreement with the aforementioned hypothesis, suggesting that it is the age-associated lack of neurotrophic factors (GH/IGF-I?) the cause of decreased neurogenesis. In fact, growth hormone prevents neuronal loss in the aged rat hippocampus (Azcoitia et al., 2005).

GH induces IGF-I expression in liver and other tissues; however it is not the only factor responsible for it. In fact, there are a number of pathological situations in which a clear divergence exists between GH production and plasma levels of IGF-I. This is the case, for instance, of Anorexia Nervosa; in these patients, while large burst of pituitary GH are released into the blood, IGF-I plasma levels are consistently lower than normal. The opposite situation can be observed in obese children; GH secretion is deficient in these children, but plasma IGF-I levels are normal and growth velocity is above the mean for age (Devesa et al., 1992). The common link between these two situations seems to be plasma glucose levels, low in Anorexia Nervosa and showing a tendency to hyperglycemia in obesity. Thus, it has been postulated that glucose is needed for the hepatic production of IGF-I; more particularly a product of intracellular metabolism of glucose, since 2 deoxiglucose (who can not be metabolized) is unable to induce hepatic IGF-I expression. Therefore, although GH usually is the factor responsible for the hepatic production of IGF-I, in terms of neural effects both hormones may follow different paths. In fact, studies in rats submitted to moderate and severe hypoxia showed that the spatial distribution of the neuroprotection conveyed by growth hormone correlates with the spatial distribution of the constitutive neural growth hormone receptor, but not with the neuroprotection offered by IGF-I treatment in this model. These results suggest that some of the neuroprotective effects of growth hormone are mediated directly through the growth hormone receptor and do not involve IGF-I induction (Scheepens et al., 2001).

The effects of the GH/IGF-I axis on cell turnover in the adult brain probably are not limited to neuronal progenitors, since IGF-I can promote proliferation of oligodendrocyte progenitor cells (OPCs) and differentiation and survival of oligodendrocytes (McMorris & McKinnon, 1996; Mason et al., 2000; Aberg et al., 2007; Pang et al., 2007), an effect also induced by EGF (Gonzalez-Perez & Alvarez-Buylla, 2011) whose expression and that of its receptor may also be induced by GH (Pan et al., 2011). Thus, it is likely that the agingrelated decline in GH/IGF-I and dependent changes in oligodendrocyte genesis and/or maturation may contribute to impaired remyelination in the central nervous system of aged individuals (Gilson & Blakemore, 1993; Shields et al., 1999; Franklin et al., 2002; Sim et al., 2002) and to a decline in normal cognitive function. In fact, GH has positive cognitive effects when given to GH-deficient patients.

In all, the system GH/IGF-I exerts both neuroprotective and regenerative effects at the CNS. In line with this a recent study in human patients describes that a high serum IGF-I during the rehabilitation phase of stroke correlates to better recovery of long-term function (Aberg et al., 2011).

Growth Hormone and Kynesitherapy for Brain Injury Recovery 437

disease of prematurity: punctate white matter lesions, and diffuse excessive high signal intensity. These appear to be more common than PVL but less significant in terms of their impact on individual neurodevelopment. They may, however, be associated with later

Most CP children often have poor linear growth during childhood, resulting in a diminished final adult height. However the number of studies in which it has been reported whether or not GH secretion is impaired in CP is quite limited. These studies reflect that GH provocative testing induced a GH deficient secretion. In a recent study it was indicated that diminished circulating IGF-1 and GH concentrations may explain why children with CP are smaller than normally growing children (Ali et al., 2007a). On the other hand, osteopenia is a common finding in children with CP, and seems to be associated with decreased IGF-1 and IGFBP3 plasma levels, usual markers of deficient GH secretion. The large percentage of CP children with GH deficiency (GHD) has been reported to be noteworthy. However, given the complexity of GH neurorregulation (Devesa et al., 1992) it seems to be logical that severe brain damage may affect a number of neurotransmitter pathways involved in GH

Other possible causes of decreased growth in CP include psychosocial deprivation and suboptimal nutritional status, but these are also involved in subnormal GH secretion

We studied whether GH secretion was affected in 46 CP children (28 males, 18 females; aged 3 to 11 years old). Our results indicated that 70% of the patients seemed to have deficient GH secretion (Devesa et al, 2010a), therefore they were candidates to benefit from GH

In few studies have the benefits of GH-replacement therapy in children with CP been reported, and most of these studies only reflect the increased growth observed during the treatment period with the hormone (Coniglio & Stevenson, 1995; Shim et al., 2004; Ali et al.,

Neuropsychological assessments have demonstrated that GHD is associated with reduced cognitive performance; specifically, in the majority of studies it has been found that GHD can lead to clinically relevant changes in memory, processing speed, attention, vocabulary, perceptual speed, spatial learning, and in reaction time tests. Cognitive dysfunction appears to be specifically related to GH deficiency; this hypothesis is supported by the positive correlations between serum IGF-I concentration and IQ, whereas poorer emotional wellbeing and reduced perceptual-motor performance are attributed to other pituitary hormone

In a recent study we described (Devesa et al., 2011) the positive effects of GH therapy together with psychomotor and cognitive stimulation in 11 children (7 males, 4 females; aged 3 to 7 years old) with CP and GHD. Cognitive performances in the children studied were assessed by using the Battelle Developmental Inventory Screenning Test (BDIST) (Newborg et al., 1988). Tests were performed at admission, two months after commencing psychomotor and cognitive stimulation and two months after adding GH replacement therapy to psychomotor and cognitive stimulation. Before admission all these CP children had been intensively stimulated, most of them since they were 1 year old. Psychomotor and cognitive stimulation were adapted to the specific needs of each patient. Psychomotor stimulation involved tasks aimed at improving tonic–postural control, laterality, breathing and relaxation, static and dynamic balance, motor coordination and dissociation, body image, oculomotor coordination, spatial and temporal orientation, and gross and fine motor

cognitive and behavioral disorders known to be common following preterm birth.

control, thus affecting the normal secretion of the hormone.

(Devesa et al., 1992).

replacement therapy.

2007b).

deficiencies.

It is likely that some of the positive effects of GH at central level are not directly exerted by the hormone but they are mediated trough the induction and release of a number of neurotrophic factors, as shown in Figure 11.

Some of the neurotrophic effects of GH *in vivo* could be mediated and/or enhanced through the induction of the expression and release of a number of neurotrophic factors. GH induces the expression of IGF-I, EGF, EPO; VEGF and FGF. The GH-effects on the induction of BDNF expression have not been tested yet but it is likely that they occur, directly or indirectly. GH induces an acute and strong release of neutrophiles from the bone marrow, then allowing the release of neurotrophic cytokines. GH has trophic effects on the gonads, thus facilitating estradiol and testosterone production. In turn, it is likely that the neurotrophic effects of these sexual steroids may be partially due to a positive effect on the pituitary GH release mediated by Ach and noradrenaline (Devesa et al., 1992).

Fig. 11. GH induction of the expression and/or release of a number of factors with known neurotrophic activity.

#### **3. Effects of GH treatment combined with kynesitherapy on the recovery of patients with brain injuries**

#### **3.1 Cerebral palsy children**

Cerebral palsy (CP) is a catastrophic acquired disease, occurring during development of the fetal or infant brain. It mainly affects the motor control centres of the developing brain, but can also affect cognitive functions, and is usually accompanied by a cohort of symptoms including lack of communication, epilepsy, and alterations in behavior.

Major causes for CP include abnormal intrauterine developments, due to fetal-maternal infections, asphyxia before birth, hypoxia during delivery, brain trauma during labor and delivery, and complications in the perinatal period. Apart from these, prematurity is responsible for 40%–50% of cases of CP. Periventricular leucomalacia (PVL) and parenchymal venous infarction complicating germinal matrix/intraventricular hemorrhage have long been recognized as the two significant white matter diseases responsible for the majority of cases of cerebral palsy in survivors of preterm birth.

However, in more recent studies using magnetic resonance imaging to assess the preterm brain, two new appearances have been documented, adding to the spectrum of white matter

It is likely that some of the positive effects of GH at central level are not directly exerted by the hormone but they are mediated trough the induction and release of a number of

Some of the neurotrophic effects of GH *in vivo* could be mediated and/or enhanced through the induction of the expression and release of a number of neurotrophic factors. GH induces the expression of IGF-I, EGF, EPO; VEGF and FGF. The GH-effects on the induction of BDNF expression have not been tested yet but it is likely that they occur, directly or indirectly. GH induces an acute and strong release of neutrophiles from the bone marrow, then allowing the release of neurotrophic cytokines. GH has trophic effects on the gonads, thus facilitating estradiol and testosterone production. In turn, it is likely that the neurotrophic effects of these sexual steroids may be partially due to a positive effect on the

Fig. 11. GH induction of the expression and/or release of a number of factors with known

**3. Effects of GH treatment combined with kynesitherapy on the recovery of**

Cerebral palsy (CP) is a catastrophic acquired disease, occurring during development of the fetal or infant brain. It mainly affects the motor control centres of the developing brain, but can also affect cognitive functions, and is usually accompanied by a cohort of symptoms

Major causes for CP include abnormal intrauterine developments, due to fetal-maternal infections, asphyxia before birth, hypoxia during delivery, brain trauma during labor and delivery, and complications in the perinatal period. Apart from these, prematurity is responsible for 40%–50% of cases of CP. Periventricular leucomalacia (PVL) and parenchymal venous infarction complicating germinal matrix/intraventricular hemorrhage have long been recognized as the two significant white matter diseases responsible for the

However, in more recent studies using magnetic resonance imaging to assess the preterm brain, two new appearances have been documented, adding to the spectrum of white matter

pituitary GH release mediated by Ach and noradrenaline (Devesa et al., 1992).

including lack of communication, epilepsy, and alterations in behavior.

majority of cases of cerebral palsy in survivors of preterm birth.

neurotrophic activity.

**patients with brain injuries 3.1 Cerebral palsy children**

neurotrophic factors, as shown in Figure 11.

disease of prematurity: punctate white matter lesions, and diffuse excessive high signal intensity. These appear to be more common than PVL but less significant in terms of their impact on individual neurodevelopment. They may, however, be associated with later cognitive and behavioral disorders known to be common following preterm birth.

Most CP children often have poor linear growth during childhood, resulting in a diminished final adult height. However the number of studies in which it has been reported whether or not GH secretion is impaired in CP is quite limited. These studies reflect that GH provocative testing induced a GH deficient secretion. In a recent study it was indicated that diminished circulating IGF-1 and GH concentrations may explain why children with CP are smaller than normally growing children (Ali et al., 2007a). On the other hand, osteopenia is a common finding in children with CP, and seems to be associated with decreased IGF-1 and IGFBP3 plasma levels, usual markers of deficient GH secretion. The large percentage of CP children with GH deficiency (GHD) has been reported to be noteworthy. However, given the complexity of GH neurorregulation (Devesa et al., 1992) it seems to be logical that severe brain damage may affect a number of neurotransmitter pathways involved in GH control, thus affecting the normal secretion of the hormone.

Other possible causes of decreased growth in CP include psychosocial deprivation and suboptimal nutritional status, but these are also involved in subnormal GH secretion (Devesa et al., 1992).

We studied whether GH secretion was affected in 46 CP children (28 males, 18 females; aged 3 to 11 years old). Our results indicated that 70% of the patients seemed to have deficient GH secretion (Devesa et al, 2010a), therefore they were candidates to benefit from GH replacement therapy.

In few studies have the benefits of GH-replacement therapy in children with CP been reported, and most of these studies only reflect the increased growth observed during the treatment period with the hormone (Coniglio & Stevenson, 1995; Shim et al., 2004; Ali et al., 2007b).

Neuropsychological assessments have demonstrated that GHD is associated with reduced cognitive performance; specifically, in the majority of studies it has been found that GHD can lead to clinically relevant changes in memory, processing speed, attention, vocabulary, perceptual speed, spatial learning, and in reaction time tests. Cognitive dysfunction appears to be specifically related to GH deficiency; this hypothesis is supported by the positive correlations between serum IGF-I concentration and IQ, whereas poorer emotional wellbeing and reduced perceptual-motor performance are attributed to other pituitary hormone deficiencies.

In a recent study we described (Devesa et al., 2011) the positive effects of GH therapy together with psychomotor and cognitive stimulation in 11 children (7 males, 4 females; aged 3 to 7 years old) with CP and GHD. Cognitive performances in the children studied were assessed by using the Battelle Developmental Inventory Screenning Test (BDIST) (Newborg et al., 1988). Tests were performed at admission, two months after commencing psychomotor and cognitive stimulation and two months after adding GH replacement therapy to psychomotor and cognitive stimulation. Before admission all these CP children had been intensively stimulated, most of them since they were 1 year old. Psychomotor and cognitive stimulation were adapted to the specific needs of each patient. Psychomotor stimulation involved tasks aimed at improving tonic–postural control, laterality, breathing and relaxation, static and dynamic balance, motor coordination and dissociation, body image, oculomotor coordination, spatial and temporal orientation, and gross and fine motor

Growth Hormone and Kynesitherapy for Brain Injury Recovery 439

According to these results it is likely that GH administration plays a key role in recovering from a brain injury, independently or not that a GH deficiency exists. In this regard, voluntary physical exercise is known to be a factor increasing neurogenesis and also enhancing learning and memory (Beckinstein et al., 2011). However few positive effects are obtained in CP chidren undergoing exhaustive daily physical work, as our data also reflect. Exercise is a powerful stimulus for endogenous growth hormone release, most likely through enhanced central NA tone (Devesa et al., 1992). It has been demonstrated that inhibiting PI3K/Akt signaling, one of the pathways by which GH acts (Costoya et al., 1999), blocks exercise-mediated enhancement of adult neurogenesis and synaptic plasticity in rats (Bruel-Jungerman et al., 2009). The possibility exists that the lack of significant improvements in CP children submitted to intensive physical work is due to an impaired GH secretion, as it seems to occur whenever a brain injury exists, independently that current

As described before, blindness is a common finding in CP children. We studied 20 children with CP occurring as a consequence of prematurity leading to Periventricular Leukomalacia (11 males, 9 females, aged 2,05 ± 1,43 years; mean ± SD of the mean) with critical impairment of vision and marked pallor of the optic nerve. They were treated with GH and visual stimulation performed with a tachistoscope (repetitive white light flashes, 100-150 ms, carried out in 10 phases lasting 1 min each one, 80 flashes/min; 5 days/week). Stimulation was performed in a dark isolated room. Visual evoked potentials (VEP) were recorded before commencing the treatment and after finishing it. Treatment lasted for 5 ± 2.65 months. The rationale for interrupting visual stimulation and GH treatment was based on clinical observations (for example, visual interaction with the environment, looking at

Results from this study (unpublished data) show that the children we studied presented severe visual deficiencies, characterized by a delayed conduction from the retina to the occipital cortex, as VEP showed, but these were corrected at the end of treatment period, as Figure 12 shows. Latencies were significatly decreased, while no significant changes were

Delayed conduction from the retina to the occipital cortex means a deficient myelination. This may be related to a delayed maturation of the CNS, but it is unlikely that this was the reason in these CP children since they suffered brain injuries produced by prematurity leading to Periventricular Leukomalacia in which cerebral white matter is consistently affected. Moreover, some of these children were older than 2 years old, a period of time from which it is considered that a brain damage is already established and significant improvements are unlikely to appear. Injuries involving the optic radiations, such as it happens in Periventricular Leukomalacia, have a bad prognosis (Hoyt, 2003) and most of

We utilized a tachistoscope as a stimulation method of both visual hemifields, but it is unlikely that visual stimulation alone could be responsible for the clinical and VEP changes observed after the treatment. Despite the fact that there was not a control group in our study and this could lead to missinterpretating the results obtained, most of these children had

A recent study describe that plasma levels of GH/IGF-I influence glial turnover in the white matter (Hua et al., 2009). It is not clear whether the maintenance of Oligodendrocyte Precursor Cells (OPCs) and oligodendrocyte turnover in the adult brain serves normal function or only provides a rapidly recruitable population of cells for myelin repair

been receiving intense stimulation previously without achieving significant results.

provocative tests do not reflect the existence of a GH deficiency.

objects or relatives, taking objects with the hands...).

these children are expected to remain visually handicapped.

observed in amplitudes.

skills. Cognitive stimulation involved tasks directed at improving interaction with the environment, communication, attention, perception, memory, reasoning, and concept learning. These therapies were carried out for 45 minutes per day, 5 days per week during 4 months. Recombinant human GH was given subcutaneously, 30 µg/kg/day, 5 days/week, during 2 months (after 2 months of psychomotor and cognitive stimulation).

Patients did not improve their psychomotor and cognitive status during the pretreatment period, during which only psychomotor and cognitive stimulation were performed. However, significant improvements in all BDIST domains were observed when GH was administered together with psychomotor and cognitive stimulation; specifically, patients improved in personal and social skills, adaptive behavior, gross motor skills and total psychomotor abilities, receptive and total communication, cognitive skills, and in the total score of the scale (p < 0.01), and in fine motor skills and expressive communication (p < 0.02). As expected, plasma IGF-1 and IGFBP3 significantly increased after GH treatment.

These results led us to conclude that the combined therapy involving GH replacement and psychomotor and cognitive stimulation is useful for the appropriate neurodevelopment of children with CP and GHD (Devesa et al., 2011). Since these children had received an intensive neurostimulation without significant improvements before being treated with GH, it seems to be clear that the hormone, and/or IGF-1 was the main factor responsible for the results obtained. Moreover, since exogenous GH combines with locally produced GH for repairing brain injuries (P. Devesa et al., 2011) it is feasible to assume that GH treatment may be used too in CP children without GHD.

In another study (Reimunde et al., 2011), we assessed the effects of growth hormone treatment (30 μg/kg/day) combined with physical rehabilitation in the recovery of gross motor function in children with GHD and CP (four males and six females, mean age 5.63 ± 2.32 years) as compared with that observed in a similar population of CP children (five males, five females, mean age 5.9 ± 2.18 years) without growth hormone deficiency treated only with physical rehabilitation for two months. The Gross Motor Function Measure (GMFM-88) and Modified Ashworth Scale were performed before commencing the treatment and after completion thereof. The GMFM-88 is a scale constructed for evaluation of change in gross motor function in children with cerebral palsy and consists of 88 items grouped into five dimensions, ie, dimension A (lying and rolling, 17 items), dimension B (sitting, 20 items), dimension C (crawling and kneeling, 14 items), dimension D (standing, 13 items) and dimension E (walking, running, and jumping, 24 items).

Scores for each dimension are expressed as a percentage of the maximum score for that dimension, adding the scores for all dimensions, and dividing by 5 to obtain the total score. The reliability, validity, and responsiveness of the GMFM-88 scores are documented for children with cerebral palsy (Palisano et al., 2000). The Modified Ashworth Scale is used for measuring spasticity in spastic patients (Bohannon & Smith MB, 1987).

In children with CP and GHD, Dimension A (p < 0.02), dimension B (p < 0.02), and dimension C (p < 0.02) of the GMFM-88, and the total score of the test (p < 0.01) significantly improved after the treatment; dimension D and dimension E did not increase, and four of five spastic patients showed a reduction in spasticity. However, in children with cerebral palsy and without growth hormone deficiency, only the total score of the test improved significantly after the treatment period. Plasma IGF-I values (previously low in GHD CP children) were similar in both groups at the end of the treatment period, indicating that growth hormone replacement therapy was responsible for the large differences observed between both groups in response to physical rehabilitation.

skills. Cognitive stimulation involved tasks directed at improving interaction with the environment, communication, attention, perception, memory, reasoning, and concept learning. These therapies were carried out for 45 minutes per day, 5 days per week during 4 months. Recombinant human GH was given subcutaneously, 30 µg/kg/day, 5 days/week,

Patients did not improve their psychomotor and cognitive status during the pretreatment period, during which only psychomotor and cognitive stimulation were performed. However, significant improvements in all BDIST domains were observed when GH was administered together with psychomotor and cognitive stimulation; specifically, patients improved in personal and social skills, adaptive behavior, gross motor skills and total psychomotor abilities, receptive and total communication, cognitive skills, and in the total score of the scale (p < 0.01), and in fine motor skills and expressive communication (p < 0.02). As expected, plasma IGF-1 and IGFBP3 significantly increased after GH treatment. These results led us to conclude that the combined therapy involving GH replacement and psychomotor and cognitive stimulation is useful for the appropriate neurodevelopment of children with CP and GHD (Devesa et al., 2011). Since these children had received an intensive neurostimulation without significant improvements before being treated with GH, it seems to be clear that the hormone, and/or IGF-1 was the main factor responsible for the results obtained. Moreover, since exogenous GH combines with locally produced GH for repairing brain injuries (P. Devesa et al., 2011) it is feasible to assume that GH treatment

In another study (Reimunde et al., 2011), we assessed the effects of growth hormone treatment (30 μg/kg/day) combined with physical rehabilitation in the recovery of gross motor function in children with GHD and CP (four males and six females, mean age 5.63 ± 2.32 years) as compared with that observed in a similar population of CP children (five males, five females, mean age 5.9 ± 2.18 years) without growth hormone deficiency treated only with physical rehabilitation for two months. The Gross Motor Function Measure (GMFM-88) and Modified Ashworth Scale were performed before commencing the treatment and after completion thereof. The GMFM-88 is a scale constructed for evaluation of change in gross motor function in children with cerebral palsy and consists of 88 items grouped into five dimensions, ie, dimension A (lying and rolling, 17 items), dimension B (sitting, 20 items), dimension C (crawling and kneeling, 14 items), dimension D (standing, 13

Scores for each dimension are expressed as a percentage of the maximum score for that dimension, adding the scores for all dimensions, and dividing by 5 to obtain the total score. The reliability, validity, and responsiveness of the GMFM-88 scores are documented for children with cerebral palsy (Palisano et al., 2000). The Modified Ashworth Scale is used for

In children with CP and GHD, Dimension A (p < 0.02), dimension B (p < 0.02), and dimension C (p < 0.02) of the GMFM-88, and the total score of the test (p < 0.01) significantly improved after the treatment; dimension D and dimension E did not increase, and four of five spastic patients showed a reduction in spasticity. However, in children with cerebral palsy and without growth hormone deficiency, only the total score of the test improved significantly after the treatment period. Plasma IGF-I values (previously low in GHD CP children) were similar in both groups at the end of the treatment period, indicating that growth hormone replacement therapy was responsible for the large differences observed

during 2 months (after 2 months of psychomotor and cognitive stimulation).

may be used too in CP children without GHD.

items) and dimension E (walking, running, and jumping, 24 items).

measuring spasticity in spastic patients (Bohannon & Smith MB, 1987).

between both groups in response to physical rehabilitation.

According to these results it is likely that GH administration plays a key role in recovering from a brain injury, independently or not that a GH deficiency exists. In this regard, voluntary physical exercise is known to be a factor increasing neurogenesis and also enhancing learning and memory (Beckinstein et al., 2011). However few positive effects are obtained in CP chidren undergoing exhaustive daily physical work, as our data also reflect. Exercise is a powerful stimulus for endogenous growth hormone release, most likely through enhanced central NA tone (Devesa et al., 1992). It has been demonstrated that inhibiting PI3K/Akt signaling, one of the pathways by which GH acts (Costoya et al., 1999), blocks exercise-mediated enhancement of adult neurogenesis and synaptic plasticity in rats (Bruel-Jungerman et al., 2009). The possibility exists that the lack of significant improvements in CP children submitted to intensive physical work is due to an impaired GH secretion, as it seems to occur whenever a brain injury exists, independently that current provocative tests do not reflect the existence of a GH deficiency.

As described before, blindness is a common finding in CP children. We studied 20 children with CP occurring as a consequence of prematurity leading to Periventricular Leukomalacia (11 males, 9 females, aged 2,05 ± 1,43 years; mean ± SD of the mean) with critical impairment of vision and marked pallor of the optic nerve. They were treated with GH and visual stimulation performed with a tachistoscope (repetitive white light flashes, 100-150 ms, carried out in 10 phases lasting 1 min each one, 80 flashes/min; 5 days/week). Stimulation was performed in a dark isolated room. Visual evoked potentials (VEP) were recorded before commencing the treatment and after finishing it. Treatment lasted for 5 ± 2.65 months. The rationale for interrupting visual stimulation and GH treatment was based on clinical observations (for example, visual interaction with the environment, looking at objects or relatives, taking objects with the hands...).

Results from this study (unpublished data) show that the children we studied presented severe visual deficiencies, characterized by a delayed conduction from the retina to the occipital cortex, as VEP showed, but these were corrected at the end of treatment period, as Figure 12 shows. Latencies were significatly decreased, while no significant changes were observed in amplitudes.

Delayed conduction from the retina to the occipital cortex means a deficient myelination. This may be related to a delayed maturation of the CNS, but it is unlikely that this was the reason in these CP children since they suffered brain injuries produced by prematurity leading to Periventricular Leukomalacia in which cerebral white matter is consistently affected. Moreover, some of these children were older than 2 years old, a period of time from which it is considered that a brain damage is already established and significant improvements are unlikely to appear. Injuries involving the optic radiations, such as it happens in Periventricular Leukomalacia, have a bad prognosis (Hoyt, 2003) and most of these children are expected to remain visually handicapped.

We utilized a tachistoscope as a stimulation method of both visual hemifields, but it is unlikely that visual stimulation alone could be responsible for the clinical and VEP changes observed after the treatment. Despite the fact that there was not a control group in our study and this could lead to missinterpretating the results obtained, most of these children had been receiving intense stimulation previously without achieving significant results.

A recent study describe that plasma levels of GH/IGF-I influence glial turnover in the white matter (Hua et al., 2009). It is not clear whether the maintenance of Oligodendrocyte Precursor Cells (OPCs) and oligodendrocyte turnover in the adult brain serves normal function or only provides a rapidly recruitable population of cells for myelin repair

Growth Hormone and Kynesitherapy for Brain Injury Recovery 441

2 weeks showed a severe encephalopathy with cystic cavities in both occipital lobes. VEP and auditory evoked potentials were isoelectric without any signs of conduction. Prognosis

After obtaining signed informed consent from the parents we commenced an intensive rehabilitation with him. Visual stimulation, auditory stimulation, sensorial and physical stimulation and GH treatment (0.04 mg/kg/day). Three months later the child was showing signs of positive evolution. There was electrical conduction from the retina to the cortex, he responded to auditory stimuli and spasticity was decreased. GH treatment lasted for 4 months, but physical and sensorial stimulation continued for 12 months. At this time the child was absolutely normal as functional physical and cognitive tests revealed. Currently, at age 17 months, he walks, he speaks, he laughs, he plays, he is like any other child of his age. No sequelae have been observed and the treatment did not produce any kind of

A new MRI taken when he was 1 year old showed that the brain was completely regenerated. Cystic cavities disappeared and occipital lobes were fully functional. Voxels based morphometry comparing both MRI indicated that a number of brain areas

Figures 13 and 14 respectively show MRI and voxels based morphometry studies in this

Upper images were taken at age 2-weeks. Notice the cavities affecting occipital lobes. Lower images

Fig. 13. Some images of MRI studies in the child described as a Case report.

were taken at age 1-year; no occipital cysts exist.

experienced a significant regeneration; this was particularly significant in thalamus.

could not be worst: blind, deaf, dumb and spastic tetraparesia.

adverse effects.

child.

following damage. Proliferation of oligodendrocyte precursors and recruitment of new, myelinating oligodendrocytes from immature precursors contribute to myelin repair following demyelinating lesions. Following demyelination, proliferation of OPCs and commitment, differentiation and survival of adult-born oligodendrocytes all appear to be targets of regulation by inflammatory cytokines and growth factors.

The figure shows changes observed in the latencies of Visual Evoked Potential waves registered in CP children before (black bars) and after (white bars) the treatment with GH and visual stimulation. \* p < 0.005 vs. pretreatment.

Fig. 12. Latencies of Visual Evoked Potential waves in blind CP children.

The GH/IGF-I system appears to play a particularly critical role in myelin repair. IGF expression is induced in multiple models of demyelination and is increased during remyelination (Fushimi & Sharabe, 2004). Treatment with IGF-I or overexpression of IGF-I in transgenic mice inhibits oligodendrocyte death during demyelination and/or enhances remyelination following demyelinating lesions (McMorris & McKinnon, 1996; Mason et al., 2000; Kumar et al., 2007).

The possibility exists that remyelination observed in our study, as showed by the improvements in VEPs latencies, might be due to increased plasma IGF-I values. This does not exclude a direct effect of GH on myelination. As described before, GH induces EGF and EGF-R expression, factors recently involved on central nervous system remyelination (Gonzalez-Perez & Alvarez-Buylla, 2011).

Thus, it is feasible to assume that GH treatment played a significant role in the visual recovery elicited by specific visual stimulation.

#### **Case report**

At age 3 weeks a CP baby was admitted for rehabilitation in our Center. He suffered a massive bleeding *in utero* that produced a 20-minute cardiac arrest. A MRI carried out at age

following damage. Proliferation of oligodendrocyte precursors and recruitment of new, myelinating oligodendrocytes from immature precursors contribute to myelin repair following demyelinating lesions. Following demyelination, proliferation of OPCs and commitment, differentiation and survival of adult-born oligodendrocytes all appear to be

The figure shows changes observed in the latencies of Visual Evoked Potential waves registered in CP children before (black bars) and after (white bars) the treatment with GH and visual stimulation. \* p <

The GH/IGF-I system appears to play a particularly critical role in myelin repair. IGF expression is induced in multiple models of demyelination and is increased during remyelination (Fushimi & Sharabe, 2004). Treatment with IGF-I or overexpression of IGF-I in transgenic mice inhibits oligodendrocyte death during demyelination and/or enhances remyelination following demyelinating lesions (McMorris & McKinnon, 1996; Mason et al.,

The possibility exists that remyelination observed in our study, as showed by the improvements in VEPs latencies, might be due to increased plasma IGF-I values. This does not exclude a direct effect of GH on myelination. As described before, GH induces EGF and EGF-R expression, factors recently involved on central nervous system remyelination

Thus, it is feasible to assume that GH treatment played a significant role in the visual

At age 3 weeks a CP baby was admitted for rehabilitation in our Center. He suffered a massive bleeding *in utero* that produced a 20-minute cardiac arrest. A MRI carried out at age

Fig. 12. Latencies of Visual Evoked Potential waves in blind CP children.

0.005 vs. pretreatment.

2000; Kumar et al., 2007).

**Case report**

(Gonzalez-Perez & Alvarez-Buylla, 2011).

recovery elicited by specific visual stimulation.

targets of regulation by inflammatory cytokines and growth factors.

2 weeks showed a severe encephalopathy with cystic cavities in both occipital lobes. VEP and auditory evoked potentials were isoelectric without any signs of conduction.

After obtaining signed informed consent from the parents we commenced an intensive rehabilitation with him. Visual stimulation, auditory stimulation, sensorial and physical stimulation and GH treatment (0.04 mg/kg/day). Three months later the child was showing signs of positive evolution. There was electrical conduction from the retina to the cortex, he responded to auditory stimuli and spasticity was decreased. GH treatment lasted for 4 months, but physical and sensorial stimulation continued for 12 months. At this time the child was absolutely normal as functional physical and cognitive tests revealed. Currently, at age 17 months, he walks, he speaks, he laughs, he plays, he is like any other child of his age. No sequelae have been observed and the treatment did not produce any kind of adverse effects.

A new MRI taken when he was 1 year old showed that the brain was completely regenerated. Cystic cavities disappeared and occipital lobes were fully functional. Voxels based morphometry comparing both MRI indicated that a number of brain areas experienced a significant regeneration; this was particularly significant in thalamus.

Figures 13 and 14 respectively show MRI and voxels based morphometry studies in this child.

Upper images were taken at age 2-weeks. Notice the cavities affecting occipital lobes. Lower images were taken at age 1-year; no occipital cysts exist.

Fig. 13. Some images of MRI studies in the child described as a Case report.

Growth Hormone and Kynesitherapy for Brain Injury Recovery 443

has been shown that cognitive disorders secondary to GHD may be reversed by GH

We studied (Reimunde et al., 2010) 20 male adult patients with TBI suffered in the previous 10 years.  Eleven of them presented GHD (mean age: 53.36 ± 17.35 years), while GH secretion

All patients received daily cognitive rehabilitation, 1 hour per day, 5 days per week during 3 months. The contents of cognitive rehabilitation were aimed to improve the spatial, temporal and personal orientation, visual, auditory and tactile discrimination and perception, body recognition, planning and execution of motor acts, all memory types and processes, oral and writing language, calculation and executive function tasks. GHD patients were given human GH (0.5 mg/day during 20 days and then 1 mg/day, 5 days per

To assess different cognitive abilities we used a widely recognized neuropsychological test battery, the Wechsler Adults Intelligence Scale (WAIS) (Wechsler, 1990). This assessment battery involves several sections that specifically measure multiple cognitive functions. WAIS test was performed before commencing the treatment and after 3 months of treatment

Results from our study demonstrate that individuals treated only with cognitive rehabilitation improved, specifically, their attentional functioning. However, the group treated with GH and cognitive rehabilitation improved their attentional functioning too, but moreover showed specific improvements in memory, understanding, associative thinking skills, information management and storing, learning, visual-motor dexterity and speed of

In addition, the GHD group reached greater improvements in aspects related with memory, understanding, associative thinking skills and information management and storing

These data correlated with previous results showing that GH-deficient subjects treated with GH experience significant improvements in concentration and attention, different memory

It has been described that memory performance is weakly related to the mean daily GH dose, but there was a stronger positive correlation between the increase in mental performance and the GH-induced rise in serum IGF-I; this phenomenon can be explained by the large inter-individual differences in GH sensitivity (Deijen et al., 1998). However, in our study, no significant differences between plasma IGF-I levels in both groups of patients were observed at the end of the treatment period. Thus, it is unlikely that the IGF-I rise

In conclusion, our study demonstrated, for the first time, the utility of a combination therapy involving GH replacement and cognitive rehabilitation in the recovery of cognitive impairments in patients who had suffered a TBI and a subsequent GHD. Our data show that the combination therapy has significantly greater effects than those achieved by carrying out only cognitive rehabilitation in patients with cognitive impairments secondary to a TBI without GHD. GH administration seems to be responsible for the results obtained. On these bases, we concluded that it woud be interesting to test whether GH administration to no GHD patients would improve cognitive functions in a number of neurological disorders

seemed to be normal in the other nine patients (mean age: 47.12 ± 14.55 years).

week, for 3 months). Control patients were given placebo.

replacement (Maruff & Falletti, 2005).

information than the control patients.

could be responsible for the results here obtained.

modalities and learning.

(Reimunde et al., 2010).

in all patients.

execution.

These images were obtained after comparing MRI studies carried out at 1-year interval in the child described as a Case report. The intensity of red colours is associated to greater regeneration.

Fig. 14. Voxels based morphometry.

To our knowledge this is the first description about complete human brain regeneration demonstrated with brain images. Brain plasticity may be responsible for achieving important functional recoveries in patients after a brain injury. In our case these recoveries were found not only by clinical and specific physical and cognitive tests, but they have been demonstrated with brain images. It is clear that the age at which we commenced the treatment of this child had a significant influence on the results obtained; brain development continues for 1 year after birth, approximately, but it also clear that physical and cognitive stimulation alone are not able for achieving a complete and functional recovery of the brain. Since the system GH/IGF-I has been involved in playing a very important role during embryonic brain development it seems that the treatment with the hormone was the main factor responsible for results achieved in this study. This supports our previous postulate about commencing GH treatments as soon as possible after a brain injury (Devesa et al., 2010a).

#### **3.2 Traumatic brain injury**

Traumatic brain injury (TBI) is an important health problem and a leading cause of death and disability worldwide, mainly in people under 40. Specifically, TBI is an important identified risk factor for cognitive deficits, such as attention, concentration, learning, memory, conceptual thinking, problem-solving or language (Vogenthaler, 1987). Recent studies have demonstrated that hypopituitarism, and particularly growth hormone deficiency (GHD), is common among survivors of TBI tested several months or years following head trauma (Popovic, 2005a; Popovic et al., 2005b). The subjects at risk are those who have suffered moderate-to-severe head trauma, although mild intensity trauma or even minor head injuries may precede hypopituitarism (Popovic et al., 2005b). Complex pituitary deficits are found in 35–40% of TBI patients; severe GHD occurs in 10–15% and partial GHD in 15% of TBI patients (Popovic et al., 2005b).

Neuropsychological assessments have demonstrated that GHD is associated with reduced cognitive performance; specifically, most of the studies indicate that GHD can lead to clinically relevant changes in memory, processing speed, attention, vocabulary, perceptual speed, spatial learning and in reaction time tests as well (van Dam, 2006; Falletti et al., 2006; Nieves-Martinez et al., 2010).

Cognitive dysfunction appears to be specifically related to GH deficiency; this hypothesis is supported by the positive correlations between serum IGF-I concentration and IQ. Thus, it

These images were obtained after comparing MRI studies carried out at 1-year interval in the child described as a Case report. The intensity of red colours is associated to greater regeneration.

To our knowledge this is the first description about complete human brain regeneration demonstrated with brain images. Brain plasticity may be responsible for achieving important functional recoveries in patients after a brain injury. In our case these recoveries were found not only by clinical and specific physical and cognitive tests, but they have been demonstrated with brain images. It is clear that the age at which we commenced the treatment of this child had a significant influence on the results obtained; brain development continues for 1 year after birth, approximately, but it also clear that physical and cognitive stimulation alone are not able for achieving a complete and functional recovery of the brain. Since the system GH/IGF-I has been involved in playing a very important role during embryonic brain development it seems that the treatment with the hormone was the main factor responsible for results achieved in this study. This supports our previous postulate about commencing GH treatments as soon as possible

Traumatic brain injury (TBI) is an important health problem and a leading cause of death and disability worldwide, mainly in people under 40. Specifically, TBI is an important identified risk factor for cognitive deficits, such as attention, concentration, learning, memory, conceptual thinking, problem-solving or language (Vogenthaler, 1987). Recent studies have demonstrated that hypopituitarism, and particularly growth hormone deficiency (GHD), is common among survivors of TBI tested several months or years following head trauma (Popovic, 2005a; Popovic et al., 2005b). The subjects at risk are those who have suffered moderate-to-severe head trauma, although mild intensity trauma or even minor head injuries may precede hypopituitarism (Popovic et al., 2005b). Complex pituitary deficits are found in 35–40% of TBI patients; severe GHD occurs in 10–15% and partial GHD

Neuropsychological assessments have demonstrated that GHD is associated with reduced cognitive performance; specifically, most of the studies indicate that GHD can lead to clinically relevant changes in memory, processing speed, attention, vocabulary, perceptual speed, spatial learning and in reaction time tests as well (van Dam, 2006; Falletti et al., 2006;

Cognitive dysfunction appears to be specifically related to GH deficiency; this hypothesis is supported by the positive correlations between serum IGF-I concentration and IQ. Thus, it

Fig. 14. Voxels based morphometry.

after a brain injury (Devesa et al., 2010a).

in 15% of TBI patients (Popovic et al., 2005b).

**3.2 Traumatic brain injury**

Nieves-Martinez et al., 2010).

has been shown that cognitive disorders secondary to GHD may be reversed by GH replacement (Maruff & Falletti, 2005).

We studied (Reimunde et al., 2010) 20 male adult patients with TBI suffered in the previous 10 years.  Eleven of them presented GHD (mean age: 53.36 ± 17.35 years), while GH secretion seemed to be normal in the other nine patients (mean age: 47.12 ± 14.55 years).

All patients received daily cognitive rehabilitation, 1 hour per day, 5 days per week during 3 months. The contents of cognitive rehabilitation were aimed to improve the spatial, temporal and personal orientation, visual, auditory and tactile discrimination and perception, body recognition, planning and execution of motor acts, all memory types and processes, oral and writing language, calculation and executive function tasks. GHD patients were given human GH (0.5 mg/day during 20 days and then 1 mg/day, 5 days per week, for 3 months). Control patients were given placebo.

To assess different cognitive abilities we used a widely recognized neuropsychological test battery, the Wechsler Adults Intelligence Scale (WAIS) (Wechsler, 1990). This assessment battery involves several sections that specifically measure multiple cognitive functions. WAIS test was performed before commencing the treatment and after 3 months of treatment in all patients.

Results from our study demonstrate that individuals treated only with cognitive rehabilitation improved, specifically, their attentional functioning. However, the group treated with GH and cognitive rehabilitation improved their attentional functioning too, but moreover showed specific improvements in memory, understanding, associative thinking skills, information management and storing, learning, visual-motor dexterity and speed of execution.

In addition, the GHD group reached greater improvements in aspects related with memory, understanding, associative thinking skills and information management and storing information than the control patients.

These data correlated with previous results showing that GH-deficient subjects treated with GH experience significant improvements in concentration and attention, different memory modalities and learning.

It has been described that memory performance is weakly related to the mean daily GH dose, but there was a stronger positive correlation between the increase in mental performance and the GH-induced rise in serum IGF-I; this phenomenon can be explained by the large inter-individual differences in GH sensitivity (Deijen et al., 1998). However, in our study, no significant differences between plasma IGF-I levels in both groups of patients were observed at the end of the treatment period. Thus, it is unlikely that the IGF-I rise could be responsible for the results here obtained.

In conclusion, our study demonstrated, for the first time, the utility of a combination therapy involving GH replacement and cognitive rehabilitation in the recovery of cognitive impairments in patients who had suffered a TBI and a subsequent GHD. Our data show that the combination therapy has significantly greater effects than those achieved by carrying out only cognitive rehabilitation in patients with cognitive impairments secondary to a TBI without GHD. GH administration seems to be responsible for the results obtained. On these bases, we concluded that it woud be interesting to test whether GH administration to no GHD patients would improve cognitive functions in a number of neurological disorders (Reimunde et al., 2010).

Growth Hormone and Kynesitherapy for Brain Injury Recovery 445

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### **4. Conclusions**

Throughout this chapter we have reviewed the evidence showing that adult neurogenesis is a physiological mechanism to repair brain damage. We analyzed a number of factors directly involved in this process.

We demonstrated that both GH and its receptor are expressed in neural precursors in mice, and we showed that after a brain injury, in rats, the treatment with the hormone increases the number of newly born stem cells produced in response to the damage. We demonstrated that GH is present in the cytoplasm of these newly born cells, most likely for facilitating their migration from neurogenic niches. Our data in cell cultures obtained from mice stem cells demonstrate that the hormone plays a key role for neural cells survival.

In human patients with brain injury and GH-deficiency, we demonstrated that GH treatment together with physical and cognitive stimulation is responsible for the recovery of motor and cognitive abilities. GH treatment also appears to play a key role for remyelination of the visual pathways in children with cerebral palsy.

For the first time we showed here that an early treatment with the hormone can recover a brain injury in the first months of the life.

Although not presented here, GH treatment may repair central neurogenic dysphagias (Devesa et al., 2009; Reimunde et al., 2011, submitted for publication).

By analyzing the role of many factors positively involved on adult neurogenesis, we postulate that some of the positive effects of GH at the central level may be due to GHinduced expression of a number of neurotrophic factors and/or to enhanced release of some of them.

We conclude that GH treatment is a powerful tool for being used in a number of brain injuries, including neurodegenerative non-genetical diseases (for example, multiple sclerosis, Alzheimer, etc). It seems that there is no need that GH-deficiency exists for the hormone being useful.

GH treatments have to be well scheduled and short in time. The positive effects of the hormone remain in the time.

#### **5. Acknowledgements**

These studies were supported by Foundation Foltra (Teo, Spain). Studies in human patients were carried out in Medical Center Proyecto Foltra (Teo, Spain). Animal studies were carried out in the Department of Physiology, School of Medicine, Santiago de Compostela, Spain, under the direction of Professor Jesús Devesa and Professor Víctor Arce. Studies in NSCs were performed in the Institute of Neurosciences of the School of Medicine of Coimbra, Portugal, under the direction of Professor Joao Malva; his collaboration is greatly appreciated. Voxel based morphometries were performed by Brain Dynamics (Málaga, Spain).

#### **6. References**

Abdipranoto A, Wu A, Stayte S, et al. (2008). The role of neurogenesis in neurodegenerative diseases and its implications for therapeutic development. *CNS Neurol Disord Drug Targets*. 7:187-210.

Throughout this chapter we have reviewed the evidence showing that adult neurogenesis is a physiological mechanism to repair brain damage. We analyzed a number of factors

We demonstrated that both GH and its receptor are expressed in neural precursors in mice, and we showed that after a brain injury, in rats, the treatment with the hormone increases the number of newly born stem cells produced in response to the damage. We demonstrated that GH is present in the cytoplasm of these newly born cells, most likely for facilitating their migration from neurogenic niches. Our data in cell cultures obtained from mice stem

In human patients with brain injury and GH-deficiency, we demonstrated that GH treatment together with physical and cognitive stimulation is responsible for the recovery of motor and cognitive abilities. GH treatment also appears to play a key role for remyelination

For the first time we showed here that an early treatment with the hormone can recover a

Although not presented here, GH treatment may repair central neurogenic dysphagias

By analyzing the role of many factors positively involved on adult neurogenesis, we postulate that some of the positive effects of GH at the central level may be due to GHinduced expression of a number of neurotrophic factors and/or to enhanced release of some

We conclude that GH treatment is a powerful tool for being used in a number of brain injuries, including neurodegenerative non-genetical diseases (for example, multiple sclerosis, Alzheimer, etc). It seems that there is no need that GH-deficiency exists for the

GH treatments have to be well scheduled and short in time. The positive effects of the

These studies were supported by Foundation Foltra (Teo, Spain). Studies in human patients were carried out in Medical Center Proyecto Foltra (Teo, Spain). Animal studies were carried out in the Department of Physiology, School of Medicine, Santiago de Compostela, Spain, under the direction of Professor Jesús Devesa and Professor Víctor Arce. Studies in NSCs were performed in the Institute of Neurosciences of the School of Medicine of Coimbra, Portugal, under the direction of Professor Joao Malva; his collaboration is greatly appreciated. Voxel based morphometries were performed by Brain Dynamics (Málaga,

Abdipranoto A, Wu A, Stayte S, et al. (2008). The role of neurogenesis in neurodegenerative

diseases and its implications for therapeutic development. *CNS Neurol Disord Drug*

cells demonstrate that the hormone plays a key role for neural cells survival.

**4. Conclusions**

of them.

Spain).

**6. References**

*Targets*. 7:187-210.

hormone being useful.

hormone remain in the time.

**5. Acknowledgements**

directly involved in this process.

Growth Hormone and Kynesitherapy for Brain Injury Recovery 447

Devesa J, Devesa P & Reimunde P. (2010b). [Growth hormone revisited]. *Med Clin (Barc)*

Devesa J, Alonso B, Casteleiro N, et al (2011). Effects of recombinant growth hormone (GH)

Devesa P, Reimunde P, Gallego R, Devesa J & Arce VM. (2011). Growth hormone (GH)

Devesa P. (2011). Effects of growth hormone on the central and peripheral neural repair.

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