Unilateral Carotid Ligation Research Articles

BDNF Protects against Spatial Memory Deficits Following Neonatal Hypoxia-Ischemia

Hypoxic-ischemic (H-I) brain injury in the human perinatal period often leads to significant long-term neurobehavioral dysfunction in the cognitive and sensory-motor domains. Using a neonatal H-I injury model (unilateral carotid ligation followed by hypoxia) in postnatal day seven rats, previous studies have shown that neurotrophins, such as brain-derived neurotrophic factor (BDNF), can be protective against neural tissue loss. The present study explored potential relationships between neural protective and behavioral protective strategies in this neonatal H-I model by determining if neonatal H-I was associated with behavioral spatial learning and memory deficits and whether the neurotrophin BDNF was protective against both brain injury and spatial learning/memory dysfunction. Postnatal day seven rats received vehicle or BDNF pretreatments (intracerebroventricular injections) followed by H-I or sham treatments and then tested for spatial learning and memory on the simple place task in the Morris water maze from postnatal days 20 to 30, and their brains were histologically analyzed at 4 weeks following treatments. H-I rats with vehicle pretreatment displayed significant tissue loss in the hippocampus (including CA1 neurons), cortex, and striatum, as well as severe spatial memory deficits (e.g., short probe times). BDNF pretreatment resulted in significant protection against both H-I-induced brain tissue losses and spatial memory impairments. These findings indicate that unilateral H-I brain injury in a neonatal rodent model is associated with cognitive deficits, and that BDNF pretreatment is protective against both brain injury and spatial memory impairment.

BDNF Blocks Caspase-3 Activation in Neonatal Hypoxia–Ischemia

Hypoxic–ischemic (H-I) injury to the brain in the perinatal period often leads to significant long-term neurological deficits. In a model of neonatal H-I injury in postnatal day 7 rats, our previous data have shown that cell death with features of apoptosis is prominent between 6 and 24 h after H-I and that neurotrophins, particularly BDNF, can markedly protect against tissue loss. During brain development, caspase-3 is required for normal levels of programmed cell death. Utilizing an antibody specific for the activated form of caspase-3, CM1, we now show that caspase-3 is specifically activated in neuronal cell bodies and their processes beginning at 6 h and peaking 24 h following unilateral carotid ligation and exposure to hypoxia in postnatal day 7 rats. Caspase-3 activation began to occur in cortex at 6 h and in striatum and hippocampus at 12–18 h. Caspase-3 activation was also observed in developing oligodendrocytes. Intracerebroventricular injection of BDNF prior to H-I injury almost completely abolished evidence of H-I-induced caspase-3 activation in vivo. Utilizing a specific molecular marker of an apoptotic pathway, these findings demonstrate that H-I injury to the developing brain is a strong apoptotic stimulus leading to caspase-3 activation, that BDNF can block this process in vivo, and that the ability of BDNF to inhibit caspase activation and subsequent apoptosis likely accounts in large part for its protection against neuronal injury in this model.

Hypoxia, hyperoxia, ischemia, and brain necrosis.

Human brains show widespread necrosis when death occurs after coma due to cardiac arrest, but not after hypoxic coma. It is unclear whether hypoxia alone can cause brain damage without ischemia. The relationship of blood oxygenation and vascular occlusion to brain necrosis is also incompletely defined. We used physiologically monitored Wistar rats to explore the relationship among arterial blood oxygen levels, ischemia, and brain necrosis. Hypoxia alone (PaO2 = 25 mm Hg), even at a blood pressure (BP) of 30 mm Hg for 15 minutes, yielded no necrotic neurons. Ischemia alone (unilateral carotid ligation) caused necrosis in 4 of 12 rats, despite a PaO2 > 100 mm Hg. To reveal interactive effects of hypoxia and ischemia, groups were studied with finely graded levels of hypoxia at a fixed BP, and with controlled variation in BP at fixed PaO2. In separate series, focal ischemic stroke was mimicked with transient middle cerebral artery (MCA) occlusion, and the effect of low, normal, and high PaO2 was studied. Quantitated neuropathology worsened with every 10 mm Hg decrement in BP, but the effect of altering PaO2 by 10 mm Hg was not as great, nor as consistent. Autoradiographic study of cerebral blood flow with 14C-iodoantipyrine revealed no hypoxic vasodilatation during ischemia. In the MCA occlusion model, milder hypoxia than in the first series (PaO2 = 46.5 +/- 1.4 mm Hg) exacerbated necrosis to 24.3 +/- 4.7% of the hemisphere from 16.6 +/- 7.0% with normoxia (PaO2 = 120.5 +/- 4.1 mm Hg), whereas hyperoxia (PaO2 = 213.9 +/- 5.8 mm Hg) mitigated hemispheric damage to 7.50 +/- 1.86%. Cortical damage was strikingly sensitive to arterial PaO2, being 12.8 +/- 3.1% of the hemisphere with hypoxia, 7.97 +/-4.63% with normoxia, and only 0.3 +/- 0.2% of the hemisphere with hyperoxia (p < 0.01), and necrosis being eliminated completely in 8 of 10 animals. Hypoxia without ischemia does not cause brain necrosis but hypoxia exacerbates ischemic necrosis. Hyperoxia potently mitigates brain damage in this MCA occlusion model, especially in neocortex.

Effect of policosanol on cerebral ischemia in Mongolian gerbils.

Policosanol is a mixture of higher aliphatic primary alcohols isolated from sugar cane wax, whose main component is octacosanol. An inhibitory effect of policosanol on platelet aggregation and cerebral ischemia in animal models has been reported. Thus, the objective of the present study was to evaluate the effect of policosanol on cerebral ischemia induced by unilateral carotid ligation and bilateral clamping and recirculation in Mongolian gerbils. Policosanol (200 mg/kg) administered immediately after unilateral carotid ligation and at 12- or 24-h intervals for 48 h significantly inhibited mortality and clinical symptoms when compared with controls, whereas lower doses (100 mg/kg) were not effective. Control animals showed swelling (tissue vacuolization) and necrosis of neurons in all areas of the brain studied (frontal cortex, hippocampus, striatum and olfactory tubercle), showing a similar injury profile. In the group treated with 200 mg/kg policosanol swelling and necrosis were significantly reduced when compared with the control group. In another experimental model, comparison between groups showed that the brain water content of control gerbils (N = 15) was significantly higher after 15 min of clamping and 4 h of recirculation than in sham-operated animals (N = 13), whereas policosanol (200 mg/kg) (N = 19) significantly reduced the edema compared with the control group, with a cerebral water content identical to that of the sham-operated animals. cAMP levels in the brain of control-ligated Mongolian gerbils (N = 8) were significantly lower than those of sham-operated animals (N = 10). The policosanol-treated group (N = 10) showed significantly higher cAMP levels (2.68 pmol/g of tissue) than the positive control (1.91 pmol/g of tissue) and similar to those of non-ligated gerbils (2.97 pmol/g of tissue). In conclusion, our results show an anti-ischemic effect of policosanol administered after induction of cerebral ischemia, in two different experimental models in Mongolian gerbils, suggesting a possible therapeutic effect in cerebral vascular disorders.

Brain injury after neonatal hypoxia–ischemia in rats: a role of cysteine?

The aim of this study was to investigate the role of cysteine in development of brain damage after hypoxia–ischemia (HI) in neonatal rats. Rat pups were subjected to unilateral carotid ligation and exposure to hypoxia (7.7% oxygen) for 60 or 90 min. A subtoxic dose of cysteine were administered before or after HI and the unilateral brain injury was evaluated 14 days after the insult and expressed as ipsilateral weight deficit as % of the contralateral hemisphere. In some experiments the changes of extracellular (e.c.) cysteine in the cerebral cortex were sampled with microdialysis and analyzed with HPLC. Cysteine in a dose of 0.2 mg/g s.c. given before 60 min of HI increased the extent of brain injury by 59%. The effect of posttreatment was limited and dependent on the duration of HI: 0.2 mg/g of cysteine given after 90 min of HI increased the degree of brain injury by 25%, whereas the same dose administered after 60 min of HI was ineffective in spite of that this combination of cysteine and HI resulted in e.c. cysteine concentrations 3–4 times higher than those observed in non-treated HI controls. These data show that subtoxic doses of cysteine administered before or after HI enhances brain injury. However, e.c. cysteine levels exceeding those induced by HI are required which makes a substantial contribution of cysteine in the pathophysiology of HI brain injury in the neonatal rat unlikely.

Flow-induced vascular remodeling in the rat carotid artery diminishes with age.

Vascular remodeling is regulated by a combination of hemodynamic, environmental, and genetic factors and may be influenced by age. To evaluate age-dependent remodeling in rats, we developed and used a quantitative highly reproducible model of carotid flow alteration. Fourteen juvenile (99+/-3 g) and 9 adult (199+/-5 g) male inbred Fischer rats underwent ligation of the left internal and external carotid arteries under anesthesia. Left common carotid blood flow immediately decreased by approximately 93%, whereas flow in the contralateral carotid increased by approximately 46%. After 4 weeks, the left carotid outer diameter (OD) significantly decreased in both juvenile and adult rats (as measured in vivo and by histological morphometry) compared with sham-operated rats. Changes in shear stress acutely mirrored the changes in blood flow. OD increased and shear stress returned to initial values after chronic exposure to increased flow in juvenile but not adult rats. To develop a simple quantitative index of remodeling that would not require killing the animals, we measured the OD in vivo and compared the ratio of right to left OD (OD ratio [ODR]) between groups. The initial ODR for all groups was approximately 1.0. After 4 weeks of altered flow, the ODR was significantly greater in juvenile than in adult rats (1.48+/-0.05 versus 1.29+/-0.04, respectively; P=.030), indicating that juvenile rats experienced more extensive remodeling than did the adult rats. We also found that unilateral carotid ligation caused a left versus right difference in endothelial NO synthase protein levels after 4 weeks that was not present in the sham-operated animals. Thus, the model described here shows that flow-induced vascular remodeling is dependent on age and supports the hypothesis that the driving force for remodeling involves shear stress and possibly NO. Because the model is quantitative, it allows dissection of the genetic factors that regulate remodeling in inbred rat strains.

Glutathione Peroxidase Activity Decreases and Hydrogen Peroxide Accumulates after Perinatal Hypoxia-Ischemia in SOD 1 Overexpressing Mice • 1714

Neonatal mice transgenic (tg) for copper/zinc-superoxide dismutase (SOD 1) were found to have greater brain injury after hypoxia-ischemia (HI) than their nontransgenic (ntg) littermates (Pediatr Res 39: 204, 1996). No upregulation of catalase activity was found in neonatal SOD tg mice versus their ntg littermates, before or after HI (Soc Neurosci Abst 22: 1430, 1996). To further assess the roles of anti-oxidant enzymes after HI, we measured glutathione peroxidase (GP) activity and hydrogen peroxide (H2O2) concentration in the brains (cortex and hippocampus) of neonatal SOD tg and ntg mice before and after HI. 15 litters (164 pups) of CD1 mice tg for SOD were identified by gel electrophoresis. On postnatal day 7, half of the pups were subjected to unilateral carotid ligation and 30 mintues of hypoxia in 8% O2. GP activity and [H2O2] were assessed in control mice (no HI), immediately after HI, and 24 hours after HI. Contrary to catalase, GP activity dropped precipitously after injury, in both SOD tg and ntg mice: from 129.4 ± 47.3 U/(mg protein) in SOD tg cortex before injury, to 15.9 ± 7.4 U/(mg protein) in SOD tg cortex 24 hours after injury (p<0.01; similar findings observed in the hippocampus and in ntg brains). Although there was a trend towards greater GP activity in ntg vs. SOD tg brains, it was not significant. Aminotriazole inhibition of catalase activity (a qualitative measure of [H2O2]), was similar between tg and ntg brains before and 2 hours after injury. However, 24 hours after injury, greater aminotriazole inhibition was observed in the tg mice vs. their ntg littermates (p<0.01), indicating greater [H2O2] in these brains. Taken together, these findings suggest that the fall in GP activity after perinatal HI leads to an accumulation of H2O2 in SOD tg brains. This could explain the increased injury after HI previously observed in these tg mice, and suggests a role for GP in the mechanism of oxidative damage after HI in the neonate. This research was supported in part by K12 NS01692-04, RO1 NS33997-01A1, NS35902, NS32553, and AG08938.

Vertical osteotomy for extending the mandibular rami in vertical posterior insufficiency

Several reports indicate that the activity of the hypothalamic–pituitary–adrenal axis (HPA) is increased after a brain insult and that its down-regulation can improve detrimental outcomes associated with ischemic brain injuries. Granulocyte-colony stimulating factor (G-CSF) is a neuroprotective drug shown in the naïve rat to regulate hormones of the HPA axis. In this study we investigate whether G-CSF confers its neuroprotective properties by influencing the HPA response after neonatal hypoxia–ischemia (HI). Following the Rice–Vannucci model, seven day old rats (P7) were subjected to unilateral carotid ligation followed by 2.5 h of hypoxia. To test our hypothesis, metyrapone was administered to inhibit the release of rodent specific glucocorticoid, corticosterone, at the adrenal level. Dexamethasone, a synthetic glucocorticoid, was administered to agonize the effects of corticosterone. Our results show that both G-CSF and metyrapone significantly reduced infarct volume while dexamethasone treatment did not reduce infarct size even when combined with G-CSF. The protective effects of G-CSF do not include blood brain barrier preservation as suggested by the brain edema results. G-CSF did not affect the pituitary released adrenocorticotropic hormone (ACTH) levels in the blood plasma at 4 h, but suppressed the increase of corticosterone in the blood. The administration of G-CSF and metyrapone increased weight gain, and significantly reduced the Bax/Bcl-2 ratio in the brain while dexamethasone reversed the effects of G-CSF. The combination of G-CSF and metyrapone significantly decreased caspase-3 protein levels in the brain, and the effect was antagonized by dexamethasone. We report that G-CSF is neuroprotective in neonatal HI by reducing infarct volume, by suppressing the HI-induced increase of the Bax/Bcl-2 ratio, and by decreasing corticosterone in the blood. Metyrapone was able to confer similar neuroprotection as G-CSF while dexamethasone reversed the effects of G-CSF. In conclusion, we show that decreasing HPA axis activity is neuroprotective after neonatal HI, which can be conferred by administering G-CSF.

Acetazolamide produced blood flow velocity changes measured by laser Doppler in gerbils with reduced CBF

The effect of acetazolamide on the cerebral blood flow was studied in gerbils with unilateral carotid ligation. According to the effect of ligation the animals were divided into three groups: first group-the reduction more than 70%, second-CBF reduction 30-70% and the third group-CBF reduction less than 30%. The effect of acetazolamide administration was closely related to the effect of carotid ligation. More reduction of CBF was produced by carotid ligation, less increase of CBF after acetazolamide injection was noticed. The acetazolamide vascular reserve test was found a sensitive and useful method for detecting even modest reduction of vascular reserve in animals with slight - less than 30% CBF decrease following carotid ligation.

The stress of being restrained reduces brain damage after a hypoxic-ischaemic insult in the 7-day-old rat.

Experimental animals and patients are immobilized to allow continuous monitoring of physiological parameters. Restraint stress affects brain neurotransmitter levels and induces expression of immediate early genes. Whether immobilization stress affects outcomes in newborn animals which have suffered a hypoxic-ischaemic insult is unknown. Twenty 7-day-old rats subjected to unilateral carotid ligation followed by 2 h hypoxia were randomly assigned to carry a rectal probe or to move freely. The 10 restrained animals showed 50% reduction in damage in all brain regions (p < 0.0.3). Plasma lactate levels, a marker of stress, were three times higher in animals carrying a rectal probe (p < 0.0.02) than those moving freely. We conclude that the stress of being restrained reduced damage after a hypoxic-ischaemic insult in the immature rat.

L-NAME Reduces Infarct Volume in a Filament Model of Transient Middle Cerebral Artery Occlusion in the Rat Pup

The importance of nitric oxide (NO) during focal cerebral ischemia remains controversial as studies have suggested both a neurotoxic and neuroprotective role. In the 7 d old rat pup, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, reduced infarct volume in a model of unilateral carotid ligation with 2.5 h exposure to 8% O2. The current study examined whether NO is neurotoxic in a filament model of transient middle cerebral artery occlusion (MCAO) in the 14-18-d-old rat pup. We developed a reproducible filament model of transient MCAO in 14-18-d-old spontaneously hypertensive rats (35 g) by passing a no. 6-0 (0.07-mm) nylon filament via the carotid artery to occlude the middle cerebral artery for 4 h under normoxic conditions. After filament removal and reperfusion for 24 h, we determined infarct volume using the mitochondrial stain 2,3,5-triphenyltetrazolium chloride. NO synthesis was inhibited using NG-nitro-L-arginine methyl ester (L-NAME) at a dose of 3 mg/kg, intraperitoneally, 1 h before MCAO. We measured infarct volume in control (n = 7) and L-NAME (n = 7) groups. L-NAME reduced infarct volume by 55% (p < 0.01). In the control group, infarct volume (180 +/- 29 mm3) averaged 49 +/- 7% of the left hemisphere (359 +/- 16 mm3). In the L-NAME-treated group, infarct volume (77 +/- 19 mm3) was 22 +/- 5% of the left hemispheric volume (344 +/- 2 mm3). These findings support earlier studies that used models of neonatal hypoxic-ischemic brain injury and suggest a neurotoxic role of NO. They extend these observations by demonstrating a significant reduction in infarct volume in a stroke model in the immature rat pup.

Reduction in cerebral ischemic injury in the newborn rat by potentiation of endogenous adenosine.

Because of ontogenic influences on the pathophysiologic mechanisms of brain injury in the perinatal brain, and in particular, the incomplete development of adenosine receptor systems, we investigated the potential for adenosine to provide cerebro-protection in a well established newborn rat model of hypoxia-ischemia. Fifteen litters of postnatal d 7 animals were subjected to unilateral carotid ligation and exposure to hypoxia (8% oxygen) for 3 h. Immediately after hypoxia-ischemia, animals received either the adenosine deaminase inhibitor deoxycoformycin (DCF; 2.5 mg/kg intraperitoneally) or the adenosine uptake inhibitor propentofylline (PPF; 10 mg/kg intraperitoneally); paired littermates received an equivalent volume of normal saline. On postnatal d 14, injury or protection was assessed by differences in hemispheric weights, morphometric determinations of infarct area, and histopathologic analyses. DCF resulted in a 34% (p = 0.02) and 31% (p = 0.03) reduction in hemispheric weight disparities and infarct area, respectively; for PPF, these reductions were 46% (p = 0.03) and 32% (p = 0.04), respectively. Light microscopic examinations of striatum, thalamus, hippocampus, and cortex revealed that both drugs significantly improved histologic scores as well. Measurements in six separate litters indicated that neither drug significantly reduced core body temperature for at least 6 h postadministration. These findings indicate that potentiation of endogenous adenosine levels in the perinatal brain can significantly ameliorate brain injury. Each of these treatment strategies was effective even when administered after the hypoxic-ischemic insult. Thus, further investigations of adenosinergic therapies are warranted in this and other perinatal models of cerebral ischemia to elucidate in detail their potential for clinical application.

Microglia activation after neonatal hypoxic-ischemia

The inflammatory response following hypoxic-ischemia (HI) in the neonate is largely unknown. Presently, the expression of microglial antigens and the β-amyloid precursor protein (APP) were studied in relation to a dendrosomatic marker of neuronal injury (microtubule associated protein II; MAP II). HI was induced in 7-day-old rats by the combined unilateral carotid ligation and hypoxia. The pups ( n = 23) were perfusion fixed 2–3 h, 24 h, 2–4 days and 14 days after HI and compared to sham-operated controls ( n = 6). Antibodies were used for detection of the major histocompatibility complex II (OX-6), major histocompatibility complex I (OX-18) and complement receptor type 3 (OX-42). APP (APP 676–695) and MAP II (monoclonal MAP II) antigens. There was a transient APP expression 2–3 h after HI. A slight increase of microglial antigens (OX-18) was seen in the white matter 2 h after HI followed by a marked increase of OX-18, OX-6, OX-42 antigens 24 h-3–4 days in most injured regions with exception of the thalamus where a delayed (14 days) microglial response was seen. The latter event was parallelled by a delayed loss of MAP II. In conclusion, intense microglial expression occurs after neonatal HI either with an acute or delayed time-course depending on brain region.

Developmental changes in the sensitivity of the neonatal rat brain to hypoxic/ischemic injury

Developmental changes in the response of the neonatal rat brain to hypoxic/ischemic injury were examined. Hypoxic/ischemic injury was produced by unilateral carotid ligation followed by exposure to hypoxia in 1-(D1), 3-(D3), 5-(D5) and 7-day-old (D7) rats. Injury was produced in most D7 animals exposed to ≥ 120 min of 7.6 or 8% oxygen after carotid ligation. The extent of neuronal injury was variable, ranging from focal neuronal death to massive infarction. In D5 and D3 animals, there was a progressive decline in the extent of neuronal injury in response to hypoxia/ischemia. In the younger animals, bilateral injury was occasionally seen. Sham-operated animals exposed to hypoxia alone had numbers of karyorrhectic neurons similar to normal control animals in all age groups. The underlying developmental changes which account for these differences are not yet known but are likely to be multiple.

Radioiodinated tracers for the evaluation of dopamine receptors in the neonatal rat brain after hypoxic-ischemic injury.

In order to evaluate in vivo single-photon emission tomography (SPET) method of assessing cerebral function after hypoxic-ischemic injury in human neonates, we studied D1 and D2 dopamine receptors in a rat model. Seven-day-old rats underwent permanent unilateral common carotid ligation followed by exposure to 8% O2. Two weeks later, in brains with no visible loss of hemispheric volume, striatal dopaminergic receptors were studied, with [125I]TISCH and [125I]IBZM for the D1 and D2 dopamine receptors, respectively. Using [125I]TISCH, we observed no modifications of D1 receptors, but in contrast, ex vivo and in vitro autoradiographic experiments showed a 40% decrease in the striatal binding of [125I]IBZM on both the ipsilateral and the contralateral side to the carotid ligation. These alterations were detected with IBZM, a D2 dopamine receptor ligand usable for SPET imaging. Therefore, exploration of D2 receptors by SPET in human neonates suffering from perinatal hypoxia-ischemia may be valuable for the diagnosis and follow-up of cerebral function damages.

Diffusion-weighted imaging differentiates ischemic tissue from traumatized tissue.

Diffusion-weighted magnetic resonance imaging (MRI) has been shown to be particularly effective in detecting early (0 to 4 hours) pathophysiological changes in localized brain regions after cerebral ischemia. The present study sought to establish whether diffusion-weighted MRI would be similarly effective in predicting outcome after traumatic brain injury. Diffusion-weighted MRI images and T2-weighted MRI images were obtained over 4 hours after either moderate fluid percussion-induced traumatic brain injury or unilateral carotid ligation in rats. Diffusion-weighted MRI images of traumatic brain injury demonstrated focal regions of image hypointensity as early as 1 hour after trauma. The relative diffusion coefficient in these hypointense regions was significantly increased (P < .005) by 4 hours after trauma compared with the noninjured hemisphere, but only in the transverse plane in the x direction. In contrast, induction of diffuse, nonfocal ischemia by unilateral carotid ligation resulted in scattered regions of hyperintensity with a significant (P < .001) decrease in relative diffusion coefficient as early as 1 hour after ligation compared with the noninjured hemisphere. This decrease exhibited no directionality. We conclude that traumatic brain injury results in an increased water diffusion distance with the directionality indicative of bulk flow of extracellular fluid toward the lateral ventricles (vasogenic edema). In contrast, the decreased water diffusion distance with no apparent directionality observed in ischemia is most likely indicative of cytotoxic edema. Diffusion-weighted MRI therefore has the potential to differentiate cases of traumatic brain injury with no focal ischemia from those instances of traumatic brain injury in which focal ischemia is a complication.

Sympathetic Activity Enhances Glucose-related Ischemic Injury in the Rat

Studies have shown that increased sympathetic activity or increased blood and brain glucose concentration worsen postischemic brain damage. The authors evaluated the interaction of plasma glucose with epinephrine and norepinephrine during incomplete cerebral ischemia in the rat using ganglionic blockade. Rats were anesthetized with 25 micrograms.kg-1.h-1 fentanyl and 70% nitrous oxide in oxygen. Ganglionic blockade was produced in 30 rats using 8 mg/kg hexamethonium intravenously. Three plasma glucose ranges, low < 150 mg/dl, moderate = 150-300 mg/dl, and high > 300 mg/dl, were produced in each group. Ischemia was induced by unilateral carotid ligation and hemorrhagic hypotension to 30 mmHg for 30 min. Plasma norepinephrine and epinephrine were measured by radioimmunoassay. Neurologic outcome was evaluated daily for 3 days after ischemia. Ganglionic blockade decreased blood pressure before the start of ischemia and plasma epinephrine and norepinephrine during ischemia (P < 0.05). Neurologic outcome was significantly worse in rats with high glucose compared with low glucose concentrations with and without ganglionic blockade (P < 0.05). Neurologic outcome and stroke-related mortality were worse in rats with increased plasma epinephrine and norepinephrine compared with rats with ganglionic blockade when plasma glucose was less than 300 mg/dl (P < 0.05). These results indicate that increased concentration of catecholamines enhance glucose-related injury during incomplete ischemia in rats.

Hypoxia and hypoxia/ischemia affect the expression of insulin-like growth factor binding protein 2 in the developing rat brain

Brain halves were collected at various time points from 21-day-old Wistar rats exposed to either a short or prolonged period of inhalational hypoxia following unilateral carotid ligation post-insult. Neuronal loss was restricted to the side of the carotid ligation. Northern blot analysis was performed for IGFBP-2 mRNA. The prolonged hypoxia decreased the expression of IGFBP-2 five hours post insult, whereas the shorter insult level showed an ( P < 0.05) increase above control. In both groups IGFBP-2 mRNA increased to peak 3–5 days post insult. After a decline at day 6, expression was again high 7–10 days after HI. In the short hypoxia group, where there was little neuronal loss, the expression of IGFBP-2 in both hemispheres followed the same pattern over time. However prolonged hypoxia induced higher IGFBP-2 expression in the ligated hemisphere where there was extensive neuronal loss. At day 5 post-insult 5/9 rats showed an additional, slightly smaller (1.4 kb vs 1.7 kb) second transcript. The different pattern of expression associated with different degrees of injury suggest that IGFBP-2 is involved in the post asphyxial response. Hypoxia itself leads to alterations in IGFBP-2 expression. Greater expression is associated with neuronal loss. These observations suggest that the IGF system contributes to neuronal rescue and/or brain repair processes.

Expression of insulin-like growth factor-binding protein 2 (IGF-BP 2) following transient hypoxia-ischemia in the infant rat brain

Hypoxia-ischemia induced by unilateral carotid ligation followed by either 15 (moderate) or 90 (severe) min exposure to 8% oxygen was associated with induction of IGF-BP 2 mRNA expression. A specific rat IGF-BP 2 cDNA probe was used to determine the IGF-BP 2 mRNA distribution in brain sections using in situ hybridization. Untreated control rats and the non-ligated hemisphere in experimental rats expressed IGF-BP 2 mRNA in the choroid plexus, meninges and more weakly in the thalamus, hippocampus and cortical layer 5. Increased expression in experimental rats was limited to regions known to have neuronal damage. Three days after the moderate insult the signal was increased in the CA1/2 region of the hippocampus and thalamus of the ligated side. Three days after the severe insult IGF-BP 2 expression was found surrounding the infarcted regions while by 5 days after severe insult the whole infarcted volume showed induction. The results suggest a role for the IGFs in the post-asphyxial response. IGF-BP 2 may alter the bio-availability of IGF 1 or 2 or modulate their actions in the area of infarction, and thus promote cerebral repair and recovery.

Accumulation of calcitonin-gene related peptide-like immunoreactivity after hypoxic-ischaemic brain injury in the infant rat

Unilateral carotid ligation in immature rats, followed by either 15 min (moderate group) or 90 min (severe group) of hypoxia were used to assess the effects of hypoxia-ischaemia (HI) on the accumulation of the neuropeptide calcitonin-gene related peptide (CGRP). Severe, but not moderate, HI produced a massive time-dependent increase in CGRP-like immunoreactivity throughout the damaged regions of the brain (neocortex, caudate-putamen, hippocampus) beginning at 24 h and maximal at 3–5 days after HI. By 11 days after HI levels appeared to have returned to baseline. The increased immunoreactivity was largely localized to presumed axon terminals contacting neurons, blood vessels and non-nerve cells. Scattered neurons in the cingulate cortex, piriform cortex and striatum also showed increased immunoreactivity in their soma. These results raise the possibility that CGRP may be involved in neuronal repair after HI in the infant brain.