AMPA receptor antagonist, YM90K, reduces infarct volume in thrombotic distal middle cerebral artery occlusion in spontaneously hypertensive rats

We examined the effects of a potent and selective antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptor, YM90K, on brain infarction using a newly developed stroke model of thrombotic distal middle cerebral artery occlusion. Male spontaneously hypertensive rats (5–7 months old) were subjected to photochemically-induced distal middle cerebral artery occlusion as previously described [Stroke 26 (1996) 333–336]. Intravenous infusion of YM90K (n=8) (5 mg/kg per h for 1 h) or the same amount of vehicle (n=8) (alkaline saline) was started 5 min after distal middle cerebral artery occlusion. Penumbral cerebral blood flow was determined with laser-Doppler flowmetry. Three days after the ischemic insult, brains were stained with 2,3,5-triphenyltetrazolium cholride and infarct volumes were determined. One hour infusion of YM90K significantly reduced infarct volume by 34% (93±23 mm³ in control group vs. 61±25 mm³ in YM90K-treated group, P=0.017). There were no significant differences in the degrees of cerebral blood flow reduction after distal middle cerebral artery occlusion between the YM90K treated and control groups. YM90K reduces infarct volume in experimental ischemia produced by photothrombotic distal middle cerebral artery occlusion in rats. The present results demonstrated beneficial effects of AMPA receptor blockade on acute ischemic stroke.     

Inhibition of Na/H exchanger reduces infarct volume of focal cerebral ischemia in rats

Activation of Na⁺/H⁺ exchanger (NHE) may have an important role in ischemic cell death by means of intracellular overload of Na⁺ and Ca²⁺. Recent evidence has suggested that inhibitors of NHE have protective effects on myocardial ischemia both in vivo and in vitro. In this study, we tested the hypothesis that FR183998, an inhibitor of NHE, reduces infarct volume produced by focal cerebral ischemia in rats. We used 20 male spontaneously hypertensive rats. Either FR183998 (1 mg/kg; n=10), or vehicle (n=10) was given intravenously to the rats and the distal middle cerebral artery of each animal was occluded using a photothrombotic technique. We measured regional cerebral blood flow using laser-Doppler flowmetry throughout the experiments. After 3 days, infarct volume was measured in each animal group. To estimate the brain edema, we also calculated the cortical volume in both hemispheres. The infarct volume in the FR183998-treated group (82±8 mm³, mean±S.E.M.) was significantly smaller than that in the control group (115±12 mm³) (P=0.034). The cortical volume of the occluded side in the FR183998-treated group (359±7 mm³) tended to be smaller than that in the control group (378±9 mm³) (P=0.116). The regional cerebral blood flow and physiological variables during ischemia were not significantly different between the two groups throughout the experiments. These results suggest that inhibition of NHE by FR183998 may have beneficial effects in reducing infarct volume and brain edema during cerebral ischemia. Thus, NHE may play an important role in the development of neuronal damage during acute cerebral ischemia.     

Hypoglycemia induced behavioural deficit and decreased GABA receptor, CREB expression in the cerebellum of streptozoticin induced diabetic rats

Intensive glycemic control during diabetes is associated with an increased incidence of hypoglycemia, which is the major barrier in blood glucose homeostasis during diabetes therapy. The CNS neurotransmitters play an important role in the regulation of glucose homeostasis. In the present study, we showed the effects of hypoglycemia in diabetic and non- diabetic rats on motor functions and alterations of GABA receptor and CREB expression in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. Scatchard analysis of [³H]GABA binding in the cerebellum of diabetic hypoglycemic and control hypoglycemic rats showed significant (P < 0.01) decrease in B_max and K_d compared to diabetic and control rats. Real-time PCR amplification of GABA receptor subunit GABA_Aα1 and GAD showed significant (P < 0.001) down-regulation in the cerebellum of hypoglycemic rats compared to diabetic and control rats. Confocal imaging study confirmed the decreased GABA receptors in hypoglycemic rats. CREB mRNA expression was down-regulated during recurrent hypoglycemia. Both diabetic and non-diabetic hypoglycemic rats showed impaired performance in grid walk test compared to diabetic and control. Impaired GABA receptor and CREB expression along with motor function deficit were more prominent in hypoglycemic rats than hyperglycemic which showed that hypoglycemia is causing more neuronal damage at molecular level. These molecular changes observed during hypo/hyperglycemia contribute to motor and learning deficits which has clinical significance in diabetes treatment.     

Cerebellar Glucose During Fasting and Acute Hyperglycemia in Nondiabetic Men and in Men with Type 1 Diabetes

In diabetic patients, proton magnetic resonance spectroscopy (¹H MRS) has revealed increased brain glucose concentration and metabolite alterations that indicate neuronal damage and glial cell activation. Cerebellum is known to be more resistant to hypoglycemia than cerebrum, but the effects of both chronic and acute hyperglycemia on the cerebellum are less well known. ¹H MRS was used to quantify brain glucose and metabolite levels in the cerebellum, cerebral cortex, cerebral white matter, and the thalamus of diabetic and nondiabetic men after an overnight fast and during a hyperglycemic normoinsulinemic clamp with blood glucose 12 mmol/l above baseline. Fasting glucose levels were twice as high in the cerebellum than in the cerebrum. During acute hyperglycemia, the cerebellar glucose concentration increased by 3.0 mmol/l, which equals that in the cortex, but is 35% more than in the thalamus and 173% more than in the white matter. Acute hyperglycemia also increased the cerebellar tissue water content by 10%. There were no differences between diabetic and nondiabetic participants. Notably, the patients with complication free type 1 diabetes showed brain metabolite alterations in the cerebral cortex and the white matter but not in the cerebellum. Our study suggests that diabetes does not alter glucose content or uptake in the cerebellum. The increase in tissue water during acute hyperglycemia may serve to protect the cerebellum from the potentially deleterious effects of the excess glucose.     

Post-traumatic brain hypothermia reduces histopathological damage following concussive brain injury in the rat

The purposes of this study were (1) to document the histopathological consequences of moderate traumatic brain injury (TBI) in anesthetized Sprague-Dawley rats, and (2) to determine whether posttraumatic brain hypothermia (30°C) would protect histopathologically. Twenty-four hours prior to TBI, the fluid percussion interface was positioned over the right cerebral cortex. On the 2nd day, fasted rats were anesthetized with 70% nitrous oxide, 1% halothane, and 30% oxygen. Under controlled physiological conditions and normothermic brain temperature (37.5°C), rats were injured with a fluid percussion pulse ranging from 1.7 to 2.2 atmospheres. In one group, brain temperature was maintained at normothermic levels for 3 h after injury. In a second group, brain temperature was reduced to 30°C at 5 min post-trauma and maintained for 3 h. Three days after TBI, brains were perfusion-fixed for routine histopathological analysis. In the normothermic group, damage at the site of impact was seen in only one of nine rats. In contrast, all normothermic animals displayed necrotic neurons within ipsilateral cortical regions lateral and remote from the impact site. Intracerebral hemorrhagic contusions were present in all rats at the gray-white interface underlying the injured cortical areas. Selective neuronal necrosis was also present within the CA3 and CA4 hippocampal subsectors and thalamus. Post-traumatic brain hypothermia significantly reduced the overall sum of necrotic cortical neurons (519±122 vs 952±130, mean ±SE, P=0.03, Kruskal-Wallis test) as well as contusion volume (0.50±0.14 vs 2.14±0.71 mm³, P=0.004). These data document a consistent pattern of histopathological vulnerability following normothermic TBI and demonstrate hypothermic protection in the post-traumatic setting.Supported by USPHS Grants NS30291 and NS27127     

Reduced myelinated nerve fibre and endoneurial capillary densities in the forearm of diabetic and non-diabetic patients with carpal tunnel syndrome

The underlying basis of carpal tunnel syndrome (CTS) and the basis of its increased incidence in diabetes are unknown. We have quantified pathology in an uncompressed nerve (posterior interosseous nerve, PIN) in the forearm between diabetic and non-diabetic patients with CTS and control subjects. In an age- and gender-matched series, 26 diabetic patients with CTS and 20 non-diabetic patients with CTS underwent biopsy of the PIN at the time of surgical carpal tunnel release. Control subjects consisted of ten PIN biopsies taken postmortem and three biopsies taken at the time of wrist surgery. We found PIN myelinated nerve fibre density significantly reduced in diabetic (mean 5,373/mm² [95% confidence interval, 4,835–5,911]) and non-diabetic (6,617/mm² [5,697–7,537]) patients with CTS compared to control subjects (9,109/mm² [7,967–10,250], P < 0.001). Furthermore, diabetic patients had a significantly lower density than non-diabetic patients (P < 0.03). Endoneurial capillary density was also reduced in diabetic (58/mm² [50–66]) and non-diabetic (67/mm² [55–78]) patients compared to control subjects (86/mm² [72–101], P < 0.02) with no difference between diabetic and non-diabetic patients with CTS. Our results suggest that a reduction in myelinated nerve fibre and capillary densities may predispose patients, particularly those with diabetes, to develop CTS.     

In Vivo Potentiation of Glutamate-Mediated Neuronal Damage after Chronic Administration of the Glycolysis Inhibitor Iodoacetate

Neuronal damage associated with cerebral ischemia and hypoglycemia might be the consequence of the extracellular accumulation of excitatory amino acids. In previous studies we showed that elevation of glutamate and aspartate extracellular levels by inhibition of its uptake in vivo is not sufficient to induce neuronal damage unless mitochondrial energy metabolism is compromised. In the present study we show that chronic systemic administration of the glycolysis inhibitor iodoacetate (25 mg/kg) induces no damage to the brain per se but enhances neuronal vulnerability to glutamate-mediated neurotoxicity in the hippocampus. Tissue injury is well protected either by antagonizing NMDA glutamate receptors with MK-801 or by administration of pyruvate, a substrate of the tricarboxylic acid cycle. In contrast to systemic treatment, local infusions through a dialysis probe of 5 mM iodoacetate into the hippocampus induced acute lesions not sensitive to MK-801. Iodoacetate intrahippocampal perfusion induced substantial increases in the extracellular levels of glutamate (3.5-fold), taurine (8.8-fold), and particularly aspartate (35-fold). Neuronal damage under this conditions occurs very rapidly as revealed by the histological analysis of animals transcardially perfused immediately after iodoacetate perfusion. Aspartate might contribute to neuronal damage since intrahippocampal administration of this amino acid (600 nmol/μl) induces extensive lesions. The present study might suggest that impairment of glucose oxidation through the glycolytic pathway in vivo facilitates glutamate neurotoxicity. Additionally, the results indicate that pyruvate might prevent as efficiently as glutamate receptor antagonists glutamate-mediated neuronal damage associated with ischemia/hypoglycemia.     

Selegiline treatment after transient global ischemia in gerbils enhances the survival of CA1 pyramidal cells in the hippocampus

Selegiline (l-deprenyl) has shown neuroprotective effects in a variety of degenerative processes. The present experiments were designed to test whether post-ischemia administered selegiline would alleviate delayed neuronal death of the gerbil hippocampal pyramidal cells following transient global ischemia. Common carotid arteries were occluded for 5 min. Saline or selegiline, 0.25 mg/kg s.c., was administered 2 h after the ischemia followed by a daily injection for either 3 or 7 days. After decapitation, the delayed death of the hippocampal CA1 pyramidal cells was assessed using Nissl-stained sections. In situ hybridization was used to reveal the expression of hsp70 mRNA 1, 3 or 7 days after the ischemia. Animals treated with selegiline for 7 days showed significantly lower damage score (scale 0–3: 0, normal; 1, <10% of the neurons damaged; 2, 10–50% damaged; 3, >50% damaged) compared to the saline-treated animals 1.73±0.18 and 2.41±0.16 (mean±S.E.M., P=0.0133), respectively. A similar trend was found in animals after the 3-day treatment: 1.68±0.32 vs. 2.06±0.25 (P>0.5). The expression of hsp70 mRNA in the CA1 pyramidal cell layer was strong still 3 days after the ischemic insult but vanished by 7 days. Densitometric measurements using ¹⁴C-plastic standards showed that the intensity of the CA1a hsp70 signal on the 3rd day correlated negatively to the cell-damage score (r=−0.72, P<0.001), suggesting that hsp70 does not serve as a quantitative marker for CA1 neuronal injury in this model. Instead, the hsp70 expression was associated with improved neuronal survival lasting often longer in selegiline-treated animals (P>0.5). The results show that a low dose of selegiline can alleviate the delayed hippocampal neuronal death in gerbils when administered 2 h after an ischemic insult.     

Neuroprotective effect of remacemide hydrochloride in focal cerebral ischemia in the cat

The neuroprotective effects of remacemide hydrochloride ((±)-2-amino-N-(1-ethyl-1,2-diphenylethyl)acetamide hydrochloride) have been assessed with permanent occlusion of one middle cerebral artery in chloralose-anesthetized cats in which key physiologic variables have been monitored throughout the post-ischemic period. An infusion of remacemide hydrochloride (278 μg/kg/min; total dose 25 mg/kg) initiated 90 min prior to middle cerebral artery occlusion and discontinued at occlusion, reduced significantly (P < 0.02) the volume of ischemic damage (from 2505 ± 454 mm³ of vehicle-treated cats to 1266 ± 54 mm³ of remacemide hydrochloride-treated cats).     

Hu23F2G, an Antibody Recognizing the Leukocyte CD11/CD18 Integrin, Reduces Injury in a Rabbit Model of Transient Focal Cerebral Ischemia

Neutrophils are known to mediate injury in acute ischemic stroke especially during reperfusion. Migration of neutrophils into regions of ischemic injury involves binding to the endothelial cell's intercellular adhesion molecule (ICAM-1) through the leukocyte integrin, CD11/CD18. We studied the potential for neuroprotection with a humanized antibody that binds to and blocks the functions of the CD11/CD18 integrin in a rabbit model of transient focal ischemia. Fifteen New Zealand White rabbits underwent transorbital occlusion of the left middle cerebral, anterior cerebral, and internal carotid arteries using aneurysm clips for 2 h, followed by 6 h of reperfusion. Treatment with a maximally saturating dose (4 mg/kg) of a humanized CD11/CD18 monoclonal antibody (Hu23F2G, ICOS Corp., Bothell, WA) (n = 8) or placebo (n = 7) was administered 20 min after occlusion and given as a single intravenous bolus. Hemispheric ischemic neuronal damage (IND) as seen on hematoxylin- and eosin-stained sections was significantly reduced in Hu23F2G-treated animals by 57% (Hu23F2G: 15 ± 6.9%; placebo: 35 ± 5%; mean ± SEM,P < 0.05,t-test). Immunohistochemical staining with neutrophil elastase confirmed the presence of neutrophils within regions of IND in control brains. Treatment with Hu23F2G resulted in marked reduction of neutrophil infiltration. (No. of neutrophils/IND area: Hu23F2G 36.1 ± 36.7 cm⁻², placebo 460.6 ± 101.8 cm⁻²,P = 0.001.) Antagonism of neutrophil migration at the level of the CD11/CD18 integrin reduces ischemic injury in experimental stroke.     

Neuroprotective effect of ischemic preconditioning via modulating the expression of cerebral miRNAs against transient cerebral ischemia in diabetic rats

Objectives: In this study, we aimed to evaluate the effect of the Ischemic preconditioning (IPreC) on the expression profile of cerebral miRNAs against stroke by induced transient middle cerebral artery occlusion (MCAo) in diabetic rats.

Methods: Eighty male Spraque Dawley rats were allocated to eight groups. In order to evaluate the expression profile of miRNAs, we induced transient MCAo seven days after STZ-induced diabetes (DM). Also we performed IPreC 72 h before transient MCAo to assess whether IPreC could have a neuroprotective effect against ischemia-reperfusion injury.

Results: The general characteristics of STZ-treated rats included reduced body weight and elevated blood glucose levels compared to non-diabetic ones. We demonstrated that miRNA expression profiles, which are determined for biological functions such as aquaporin 4 formation (miR-29b-2, miR-124a-3p, miR-130a, miR-223 and miR-320a), glutamate toxicity (miR107, miR-145, miR-223), salvageable ischemic area (miR-9a, miR-19b, miR-29b-2, miR-341, miR-339–5p, miR-15–5p, miR-99b-5p), and neoangiogenesis (let-7f-5p, miR-126a and miR-322–3p), were regulated following IPreC. Ischemic preconditioning before cerebral ischemia significantly reduced infarction size compared with the other groups [IPreC + MCAo (27 ± 11 mm³) vs. MCAo (109 ± 15 mm³) p < 0.001; DM + IPreC + MCAo (38 ± 9 mm³) vs. DM + MCAo (165 ± 41 mm³) p < 0.001, respectively].

Discussion: The study results revealed the neuroprotective effects of ischemic preconditioning, supported with the upregulated pro-survival miRNAs in MCA infarcts.     

Microvascular damage following experimental sinus-vein thrombosis in rats

We evaluated effects of thrombosis of the superior sagittal sinus, its bridging and cortical veins (SVT) on the cerebral microvasculature in rats. Cryosections of brains (n=7) were examined for venous infarction and microvascular basal lamina damage 3 h after SVT by immunohistochemical staining of microtubule-associated protein 2 and collagen type IV. Microvessels in the infarctions showed a decrease in the number (23.5±6.1%, P<0.002) and the total area (24.9±6.5%, P<0.011) of collagen type IV-positive vessels in contrast to control areas (21.7±12.4%, P<0.007; and 26.3±15.1%, P<0.026 in contrast to control areas of unoperated animals). This study showed a significant alteration of the cerebral microvasculature in SVT, which might contribute to edema and hemorrhagic transformation.     

A new method for focal transient cerebral ischaemia by distal compression of the middle cerebral artery

A. Morancho, L. García‐Bonilla, V. Barceló, D. Giralt, M. Campos‐Martorell, S. Garcia, J. Montaner and A. Rosell (2012) Neuropathology and Applied Neurobiology 38, 617–627 A new method for focal transient cerebral ischaemia by distal compression of the middle cerebral artery Aims: Rodent experimental models are essential for in vivo study of stroke. Our aim was to develop a reproducible method of mouse transient focal cerebral ischaemia by distal artery compression. Methods: The distal middle cerebral artery (dMCA) was occluded by compression with a blunted needle, and cerebral blood flow was monitored by laser Doppler flowmetry to ensure appropriate occlusion and reperfusion in Balb/c mice. The ischaemic lesion was evaluated 24 h after occlusion by TTC staining and immunolabelling (NeuN, CD31, GFAP and Iba‐1) while the established permanent dMCA occlusion (dMCAO) model was used as a control. The corner test was performed to evaluate neurological behaviour. Results: Laser Doppler flowmetry register showed a homogenous arterial occlusion among animals. Forty‐five minutes of arterial occlusion did not lead brain infarction when evaluated by TTC staining 24 h after occlusion. Extending the cerebral ischaemia period to 60 min induced a cortically localized homogeneous brain infarct. No differences in infarct volume were detected between animals submitted to permanent or 60‐min transient dMCAO (42.33 ± 9.88 mm³ and 37.63 ± 12.09 mm³ respectively). The ischaemic injury was confirmed by immunohistochemistry in the 60‐min transient dMCAO model but not in the 45‐min model. Neurological deficits assessed with the corner test were significant only during the first 48 h but not at long term. Conclusions: This work shows an easy‐to‐perform method for the induction of brain ischaemia and reperfusion to assess stroke repair and treatment screening, with cortically localized ischaemic cell damage, low mortality and neurological impairment in the acute phase.     

3-n-butylphthalide (NBP) attenuated neuronal autophagy and amyloid-beta expression in diabetic mice subjected to brain ischemia

Abstract

Objective: The aim of this study was to investigate the protective effect of dl-3-n-butylphthalide (NBP) on brain damage in streptozotocin (STZ)-induced diabetic mice subjected to cerebral ischemia.

Methods: we pretreated diabetic mice with NBP orally for 4 weeks prior and 2 days after transient common carotid artery occlusion (CCAO) operation. Immunohistochemistry and transmission electron microscopy were performed to investigate the neuronal loss, astrocytes activation, amyloid-beta (Abeta) protein expression, and autophagy activation.

Results: The results showed that diabetes increased stroke-induced neuronal loss, astrocytes activation, Abeta generation, and autophagy activity, while NBP administration attenuated these changes. Immunofluorescence double staining of Abeta with autophagosome-specific antibody LC3 showed that most elevated Abeta⁺ signal was co-localized with LC3⁺ signal.

Conclusion: Our finding suggests that NBP attenuates Abeta generation promoted by diabetes in ischemia might act through inhibiting abnormally activated neuronal autophagy. Therefore, treatment with NBP to modulate autophagy might provide a novel therapeutic strategy for diabetes by preventing ischemic brain damage and depressing the risk of post-stroke dementia.     

A reversible component of cerebral injury as identified by the histochemical stain 2,3,5-triphenyltetrazolium chloride (TTC)

Summary The extent of histochemical change following middle cerebral artery occlusion was quantitatively determined in three groups of Sprague-Dawley rats with 2,3,5-triphenyltetrazolium chloride (a marker of mitochondrial oxidative enzyme function). In group I (n=7) occlusion was maintained for 3 h, with immediate sacrifice. In group II (n=7) occlusion was maintained for 5 h, with immediate sacrifice. In group III (n=7) occlusion was maintained for 3 h, followed by a 2-h period of reperfusion prior to sacrifice. The area of injury was significantly larger (P<0.05) in the 5-h occlusion group [15±4% (mean±SD)] compared to the 3-h occlusion group (9±2%); indicating a time-dependent worsening of the histochemical detection of injury. However, the area of injury was significantly less in the reperfusion group (5±2%) compared to the group that was evaluated after 3 h of occlusion without reperfusion (9±2%); indicating that some component of the injury revealed by 2,3,5-triphenyltetrazolium chloride is potentially reversible. These data suggest that contrary to previous understanding, the histochemical abnormality revealed by 2,3,5-triphenyltetrazolium chloride is reversible in some circumstances and does not necessarily represent inevitable infarction.Supported in part by a Research Starter Grant from the American Society of Anesthesiologists     

Effects of constraint-induced movement therapy on neurogenesis and functional recovery after early hypoxic-ischemic injury in mice

Aim Constraint‐induced movement therapy (CIMT) has emerged as a promising therapeutic strategy for improving affected upper limb function in children with hemiplegic cerebral palsy (CP). However, little is known about the changes in the brain that are induced by CIMT. This study was designed to investigate these changes and behavioural performance after CIMT intervention in mice with neonatal hypoxic‐ischemic brain injury. Method We utilized the neonatal hypoxic‐ischemic brain injury model established in mice pups. Three weeks after the injury, the mice were randomly assigned to the following three groups: the control group (n=15), the enriched‐environment group (n=17), and the CIMT with an enriched‐environment group (CIMT‐EE, n=15). 5‐bromo‐2‐deoxyuridine (BrdU) was injected daily to label proliferating cells during the 2 weeks of intervention. Results The CIMT‐EE group showed better fall rate in the horizontal ladder rung walking test (mean 5.4%, SD 3.6%) than either the control (mean 14.3%, SD 7.3%; p=0.001) or enriched‐environment (mean 12.4%, SD 7.7%; p=0.010) groups 2 weeks after the end of intervention. The CIMT‐EE group also showed more neurogenesis (mean 7069 cells/mm³, SD 4017 cells/mm³) than either the control group (mean 1555 cells/mm³, SD 1422 cells/mm³; p<0.001) or enriched‐environment group (mean 2994 cells/mm³, SD 3498 cells/mm³; p=0.001) in the subventricular zone. In the striatum, neurogenesis in the CIMT‐EE group (mean 534 cells/mm³, SD 441 cells/mm³) was greater than in the control group (mean 95 cells/mm³, SD 133 cells/mm³; p=0.001). Interpretation There was CIMT‐EE enhanced neurogenesis in the brain along with functional benefits in mice after early hypoxic‐ischemic brain injury. This is the first study to demonstrate the effects of CIMT on neurogenesis and functional recovery after experimental injury to an immature brain.     

Evaluation of CA1 damage using single-voxel H-MRS and un-biased stereology: Can non-invasive measures of N-acetyl-asparate following global ischemia be used as a reliable measure of neuronal damage?

Global brain ischemia provoked by transient occlusion of the carotid arteries (2VO) in gerbils results in a severe loss of neurons in the hippocampal CA1 region. We measured the concentration of the neuron specific N-acetyl-aspartate, [NAA], in the gerbil dorsal hippocampus by proton MR spectroscopy (¹H-MRS) in situ, and HPLC, 4 days after global ischemia. The [NAA] was correlated with graded hippocampus damage scoring and stereologically determined neuronal density. A basal hippocampal [NAA] of 8.37±0.10 and 9.81±0.44 mmol/l were found from HPLC and ¹H-MRS, respectively. HPLC measurements of [NAA] obtained from hippocampus 4 days after 2VO showed a 20% reduction in the [NAA] following 4 min of ischemia (P<0.001). ¹H-MRS measurements on gerbils subjected to 4 or 8 min of ischemia showed a similar 24% decline in the [NAA] (P<0.05). Thus, there was correlation between the HPLC and ¹H-MRS determined NAA decline. There was also a significant correlation between ¹H-MRS [NAA] and the corresponding reduction in CA1 neuronal density (P<0.004). In summary our findings show that single voxel ¹H-MRS can be used as a supplement to histological evaluation of neuronal injury in studies after global brain ischemia. Accordingly, volume selective spectroscopy has a potential for assessment of neuroprotective therapeutic compounds/strategies with respect to neuronal rescue for delayed ischemic brain damage.     

Neonatal sensory deprivation induces selective changes in the quantitative distribution of GABA-immunoreactive neurons in the rat barrel field cortex

Modified activity of the rat vibrissae from birth to adulthood induces profound alterations of the responsiveness and selectivity of neurons in the contralateral somatosensory barrel field cortex of adult rate. Because these functional properties are under the control of the intracortical inhibitory mechanisms, we investigated the effects of unilateral removal of face pad vibrissae on the quantitative distribution of intracortical γ-aminobutyric acid (GABA)-immunoreactive neurons in the rat contralateral and ipsilateral barrel field cortices. This distribution was then compared to a population of control animals. For the entire cortical depth, no significant changes in the density (7,700/mm³ vs. 7,400/mm³), proportion (13.6% vs. 14.4%), or size (11.7 μm vs. 12.5 μm) of GABA-immunoreactive neurons were found in the left contralateral vs. the right ipsilateral barrel field cortex. However, in cortical layer IV, contralateral to the deprivation, the density and proportion of GABA-immunoreactive neurons were lower (6,300/mm³ vs. 13,900/mm³, 6.0% vs. 13.6%; P < 0.01), and these neurons were larger (mean projected height of 15.1 μm vs. 10.8 μm; P < 0.01) than in the ipsilateral barrel field cortex, suggesting a specific loss of GABA expression in a subpopulation of small intracortical neurons. In addition to changes in the contralateral layer IV, GABA-immunoreactive neurons located in the ipsilateral granular layer were also affected. Indeed, their numerical density (13,900/mm³) and proportion (13.6%) were higher (P < 0.01) than in both hemispheres of control animals (average of 10,050/mm³ and 9.4%). On the other hand, GABA-immunoreactive neurons in the ipsilateral layer V were less numerous (5,600/mm³, 15.0%) than in both sides of the controls (average of 10,300/mm³, 22.0%; P < 0.01). Thus, our results show that a unilateral sensory deprivation induces highly selective changes in the intracortical GABA inhibitory circuitry of both hemispheres. These changes are located directly at the input of thalamic afferents and at an output layer of corticofugal and commissural axons and could result in a profound reorganization of the excitatory and inhibitory drives of both sides of the sensory-deprived barrel field cortex. © 1995 Wiley-Liss, Inc.