a common cause of brain injury during the perinatal period is hypoxic - ischemic injury , which frequently results in the chronic handicapping conditions such as cerebral palsy , mental retardation , learning disability , and epilepsy ( 1 ) . predicting the outcome of neonates with hypoxic - ischemic injury , however , is difficult , because some methods that predict outcome have not reliably or consistently predicted long term neurologic outcomes . recently , h - magnetic resonance spectroscopy ( mrs ) has been used as a quantitative noninvasive tool to assess the biomechanical changes associated with central nervous system injury . h - mrs , which provides objective extent of hypoxic - ischemic insults , enhances predictability and provides a method to assess treatment effect ( 2 - 5 ) . the exact mechanism by which hypoxic - ischemic brain injury occurs in neonates is not clear , although increasing evidences indicate that hypoxic - ischemic induced neuronal death includes both necrosis and apoptosis . necrosis may predominate in acute damage , whereas apoptotic injury may take time to develop . therefore , blocking the apoptotic cascade may prolong the therapeutic window after hypoxic - ischemic injury ( 6 - 8 ) . there is evidence for involvement of multiple caspases in hypoxic - ischemic brain injury . and caspase inhibitors which were developed as antiapoptotic agents , are believed to play a key role in the delayed neuronal cell death observed after hypoxic - ischemic injury ( 7 , 9 ) . the effects of growth hormone ( gh ) on the central nervous system have become more apparent in the past decade . not only it is involved in brain growth and development , but its qualities as a neuroprotective factor against injury are now appreciated . recent studies have demonstrated that gh is involved in neuroprotection during hypoxic - ischemic brain injury ( 10 , 11 ) . these protective roles are supported by the ability of gh to accelerate glial cell division and myelinogenesis , and gh is also thought to have neuroprotective roles in neurogenesis ( 11 ) . although this neuroprotective mechanism is not completely known , is probably achieved by inhibiting of caspase activities ( 12 , 13 ) . the lipid peak in the h - mr spectrum has been reported to be a marker for apoptosis during hypoxic - ischemic injury ( 14 ) . we have therefore used h - mrs to evaluate the effects of gh as a caspase inhibitor on hypoxic - ischemic injury in neonatal rat brains . the right common carotid arteries of 7-day old sprague - dawley rats ( mean weight=13.3 g ) were ligated under halothane anesthesia . after a recovery period of 3 hr gh ( eutrophin , lgphd , korea ) was administered just prior to hypoxic - ischemic insult . the rats were divided into four groups : control ( 10 l distilled water , n=29 ) , intracerebroventricular ( icv , 10 l gh in 10 l distilled water , n=23 ) , intracerebroventricular / intraperitoneal ( icv+ip , n=21 ) , and intraperitoneal ( ip , 10 mg / kg gh in distilled water , n=23 ) . localized in vivo h - mrs was performed on a bruker biospec 4.7 t mri / mrs system equipped with active shielded gradients and aspect3000 computer with tomikon hardware and software ( bruker , fallanden , switzerland ) . spectra were acquired in the right cerbral hemisphere of rats 24 hr after the onset of hypoxic - ischemic insult . water suppressed h - mr spectra were acquired using a vosy sequence with detection of the double - refocused spin echo signal from the selected voxel ( 322 l , 12 l ) using the following acquisition parameters : sw=5,000 hz , si=4,096 pts , ns=128 , tr / te=3,000/30 and 135 msec . to identify the peak at 1.3 ppm , the spectra were acquired at echo times of 30 and 135 msec in order to differentiate the lactate peak from the lipid peak . peak areas were measured and the lipid / n - acetyl aspartate ( naa ) and lipid / creatine ( cr ) ratios were used as apoptotic markers . after the h - mrs examinations on the 1st day , 6 brains from each group were perfused with 0.9% saline solution mixed with 2 units / ml of heparin , followed by perfusion with 4% paraformaldehyde in pbs solution . each brain was isolated , and tunel staining was performed using an in situ cell death detection kit , pod ( boehringer mannheim , germany ) , as described . apoptotic cells were counted 3 times in the parietal area of the brain using a 200 lens , and the mean apoptotic cell numbers were calculated using image analyzer software . gross morphologic changes were scored at 2 weeks using a 5 point grading system as method by palmer et al . ( 15 ) , where 0 indicates no change and 4 indicates the most severe injury . significance was assessed by unpaired t - test and anova followed by kruskall wallis test . spearman correlation was used to investigate relationships between lipid / naa and lipid / cr ratio and morphologic score . the right common carotid arteries of 7-day old sprague - dawley rats ( mean weight=13.3 g ) were ligated under halothane anesthesia . after a recovery period of 3 hr gh ( eutrophin , lgphd , korea ) was administered just prior to hypoxic - ischemic insult . the rats were divided into four groups : control ( 10 l distilled water , n=29 ) , intracerebroventricular ( icv , 10 l gh in 10 l distilled water , n=23 ) , intracerebroventricular / intraperitoneal ( icv+ip , n=21 ) , and intraperitoneal ( ip , 10 mg / kg gh in distilled water , n=23 ) . localized in vivo h - mrs was performed on a bruker biospec 4.7 t mri / mrs system equipped with active shielded gradients and aspect3000 computer with tomikon hardware and software ( bruker , fallanden , switzerland ) . spectra were acquired in the right cerbral hemisphere of rats 24 hr after the onset of hypoxic - ischemic insult . water suppressed h - mr spectra were acquired using a vosy sequence with detection of the double - refocused spin echo signal from the selected voxel ( 322 l , 12 l ) using the following acquisition parameters : sw=5,000 hz , si=4,096 pts , ns=128 , tr / te=3,000/30 and 135 msec . to identify the peak at 1.3 ppm , the spectra were acquired at echo times of 30 and 135 msec in order to differentiate the lactate peak from the lipid peak . peak areas were measured and the lipid / n - acetyl aspartate ( naa ) and lipid / creatine ( cr ) ratios were used as apoptotic markers . after the h - mrs examinations on the 1st day , 6 brains from each group were perfused with 0.9% saline solution mixed with 2 units / ml of heparin , followed by perfusion with 4% paraformaldehyde in pbs solution . each brain was isolated , and tunel staining was performed using an in situ cell death detection kit , pod ( boehringer mannheim , germany ) , as described . apoptotic cells were counted 3 times in the parietal area of the brain using a 200 lens , and the mean apoptotic cell numbers were calculated using image analyzer software . gross morphologic changes were scored at 2 weeks using a 5 point grading system as method by palmer et al . ( 15 ) , where 0 indicates no change and 4 indicates the most severe injury . significance was assessed by unpaired t - test and anova followed by kruskall wallis test . spearman correlation was used to investigate relationships between lipid / naa and lipid / cr ratio and morphologic score . the lipid / naa ratio was significantly lower in the icv ( 8.53.1 ) and icv / ip ( 9.22.5 ) groups than in the control group ( 12.24.6 ) ; although the lipid / naa ratio in the ip group ( 11.43.7 ) was lower than in the control group , this difference was not significant ( fig . the lipid / cr ratio was also significantly lower in the icv group ( 8.13.5 ) than in the control group ( 10.94.4 ) ; the lipid / cr ratio was lower in the icv / ip ( 9.23.4 ) and ip ( 9.63.5 ) groups than in the control group , but these differences were not statistically significant ( fig . although the number of tunel positive cells did not differ between the control and ip groups , there were fewer in the icv and icv / ip groups ( fig . we found that the morphologic scores were significantly lower in the icv group ( 1.41.3 ) and somewhat lower in the icv / ip group ( 1.81.4 ) than in the control group ( 2.21.4 ) , but not in the ip group ( 2.21.2 ) ( fig . morphologic scores significantly correlated with the lipid / naa and lipid / cr ratios ( fig . 5 ) . recently h - mrs has been used as a quantitative noninvasive assessment tool in monitoring of brain development and in the diagnosis of neurologically damaged infants ( 3 - 5 ) . h - mrs can detect metabolites such as naa and other acetyl compounds , which serve as primarily neuronal markers ; cr , including phosphocreatine and cr , which are bioenergetic markers ; choline - containing compounds ( cho ) , which are released during membrane disruption ; and lactate ( lac ) , which accumulates in response to anaerobic tissue metabolism ( 2 , 16 ) . decreased naa / cho and naa / cr ratio and increased cho / cr ratio have were in asphyxiated neonates with poor neurologic outcomes after 1 yr ( 4 , 17 ) . h - mrs in asphyxiated neonates has also shown increased lactate and decreased naa in thalamus , as well as increased lac and decreased cr in basal ganglia ( 18 , 19 ) . during hypoxic - ischemic injury , there is a significant increase in the lipid peak , which correlates with apoptotic cell death , as well as in the intensity of the lipid peak , which is directly related to the apoptotic cell count ( 14 ) . we therefore used the lipid / naa and lipid / cr ratios as apoptotic markers . newborn infants subjected to transient hypoxic - ischemic injury during birth asphyxia are apparently relatively normal soon after resuscitation but show evidence of delayed cerebral injury hours later , the magnitude of which predicts the severity of later neurodevelopmental impairment ( 20 , 21 ) . the mechanism of delayed injury is unclear , but apoptotic cells are detected in brains of infants who died after birth asphyxia , suggesting that inappropriate activation of the apoptotic pathway accounts , at least in part , for the delayed cell death ( 6 , 8 , 21 ) . apoptosis was first described as a type of cell death distinct from necrosis , with no swelling or loss of membrane integrity , and no inflammatory response from the host tissue ( 22 ) . apoptotic cells undergo a ubiquitous physiologic process that is essential to the development and survival of multicellular organisms . this process takes place during embryologic development , turnover of gastrointestinal epithelium , and the regulation of the immune system . many pathological events that cause necrosis , including hypoxic - ischemic injury , can also induce apoptosis ( 2 , 4 , 6 , 23 ) . necrosis may predominate in more intense ischemic damage , whereas apoptosis may occur during milder ischemic damage and may take time to develop ( 24 ) . immature cortical neurons have been shown to be more susceptible to apoptosis than mature neurons , perhaps because cells of younger animals more readily undergo apoptosis than cells of more mature animals ( 21 , 25 ) . thus , blocking the apoptotic cascade may prolong the therapeutic window after hypoxic - ischemic events , especially in the developing brain ( 6 - 8 ) . the ability of specific therapeutic agents to reduce caspase inhibitors , which have antiapoptotic activity and are believed to play a key role in the delayed neuronal cell death after hypoxic - ischemic injury ( 7 , 9 ) . caspase-3 is a terminal enzyme in the caspase family that activates an endonuclease ( caspase - activated dnase ) , resulting in dna fragmentation ( 26 ) . caspase inhibitors , including inhibitors of caspase-3 , may prolong the therapeutic window after hypoxic - ischemic injury ( 7 , 27 , 28 ) . recently , gh administration has been reported to inhibit neuronal death during hypoxic - ischemic injury , and to have a neuroprotective effect in the cerebral cortex , hippocampus , and thalamus ( 10 , 11 ) . although the mechanism is not completely known , several reports suggest that hypoxic - ischemic injury induces neuronal death by downregulating bcl-2 protein levels , followed by sequential activation of the caspases , and that gh protects neuronal cells by inhibiting alterations in bcl-2 protein levels and caspase activities ( 12 , 13 ) . we found that the lipid / naa ratio was significantly lower in rats administered gh by the icv and icv / ip routes , that lipid / cr ratio was significantly lower in rats administered gh by the icv , and that the degree of morphologic changes in the brain was significantly correlated with the lipid / naa and lipid / cr ratios . in our results , lipid / naa ratio and lipid / cr ratio were not significantly changed by ip administration of gh . because gh does not usually cross the blood - brain barrier , ip administration of gh taken together , these findings suggest that gh exerts neuroprotective effects in cerebral hypoxic - ischemic injury by inhibiting apoptosis , especially in the early stage after insult . our results also suggest that gh , as a caspase inhibitor , can have therapeutic value in neuroprotective effect of hypoxic - ischemic brain injury .
using 1h - mrs , we evaluated the effects of growth hormone ( gh ) as a caspase inhibitor on hypoxic - ischemic injury in neonatal rat brains . 
 the right common carotid arteries of rats were ligated , allowed to recover for 3 hr , and exposed to 8% oxygen for 2 hr . 
 gh was given just prior to hi insult and animals were divided into four groups : control , intracerebroventricular ( icv ) , intracerebroventricular / intraperitoneal ( icv / ip ) , and intraperitoneal ( ip ) . localized in vivo 1h - mrs and tunel staining 
 were performed 24 hr after hi injury . 
 lipid / n - acetyl aspartate ( naa ) and lipid / creatine ( cr ) ratios were used as apoptotic markers . 
 gross morphologic changes at 2 weeks were used to evaluate the effects of gh . 
 the lipid / naa ratio was lower in the icv and icv / ip groups than in the control , and the lipid / cr ratio was lower in the icv group than in the control . 
 the number of tunel positive cells was decreased in the icv and icv / ip groups , and the degree of morphologic change indicative of brain injury was lower in the icv group and somewhat lower in the icv / ip group . 
 the degree of morphologic change correlated with the lipid / naa and lipid / cr ratios . 
 these findings suggest that gh exerts neuroprotective effects in cerebral hypoxic - ischemic injury .