Cresyl Violet-stained Sections Research Articles

Coronal brain atlas in stereotaxic coordinates of the African spiny mouse, Acomys cahirinus.

The African spiny mouse (Acomys cahirinus) is an emerging model of mammalian epimorphic regeneration that has aroused the interest of the scientific community in the last decade. To date, studies on brain repair have been hindered by the lack of knowledge on the neuroanatomy of this species. Here, we present a coronal brain atlas in stereotaxic coordinates, which allows for three-dimensional identification and localization of the brain structures of this species. The brain of 12-week-old spiny mice was mapped in stereotaxic coordinates using cresyl violet-stained brain sections obtained from coronal cryosectioning of the brain after transcardial perfusion with fixative. The atlas is presented in 42 plates representing sections spaced 240 μm apart. Stereotaxic coordinates were validated using both a model of Parkinsonian lesion of the striatum with 6-hydroxydopamine and labeling of the corticospinal tract in the spiny mouse spinal cord using AAV1/2-GFP intracortical injections. This work presents a new tool in A. cahirinus neurobiology and opens new avenues of research for the investigation of the regenerative ability of A. cahirinus in models of brain disorders.

The connections of the insular VEN area in great apes: A histologically-guided ex vivo diffusion tractography study.

We mapped the connections of the insular von Economo neuron (VEN) area in ex vivo brains of a bonobo, an orangutan and two gorillas with high angular resolution diffusion MRI imaging acquired in 36 h imaging sessions for each brain. The apes died of natural causes without neurological disorders. The localization of the insular VEN area was based on cresyl violet-stained histological sections from each brain that were coregistered with structural and diffusion images from the same individuals. Diffusion MRI tractography showed that the insular VEN area is connected with olfactory, gustatory, visual and other sensory systems, as well as systems for the mediation of appetite, reward, aversion and motivation. The insular VEN area in apes is most strongly connected with frontopolar cortex, which could support their capacity to choose voluntarily among alternative courses of action particularly in exploring for food resources. The frontopolar cortex may also support their capacity to take note of potential resources for harvesting in the future (prospective memory). All of these faculties may support insight and volitional choice when contemplating courses of action as opposed to rule-based decision-making.

Treatment with the Ketone Body D-β-hydroxybutyrate Attenuates Autophagy Activated by NMDA and Reduces Excitotoxic Neuronal Damage in the Rat Striatum In Vivo.

The ketone bodies (KB), β-hydroxybutyrate (BHB) and acetoacetate, have been proposed for the treatment of acute and chronic neurological disorders, however, the molecular mechanisms involved in KB protection are not well understood. KB can substitute for glucose and support mitochondrial metabolism increasing cell survival. We have reported that the D-isomer of BHB (D-BHB) stimulates autophagic degradation during glucose deprivation in cultured neurons increasing cell viability. Autophagy is a lysosomal degradation process of damaged proteins and organelles activated during nutrient deprivation to obtain building blocks and energy. However, impaired or excessive autophagy can contribute to neuronal death. The aim of the present study was to test whether D-BHB can preserve autophagic function in an in vivo model of excitotoxic damage induced by the administration of the glutamate receptor agonist, N-methyl-Daspartate (NMDA), in the rat striatum. D-BHB was administered through an intravenous injection followed by either an intraperitoneal injection (i.v+i.p) or a continuous epidural infusion (i.v+pump), or through a continuous infusion of D-BHB alone. Changes in the autophagy proteins ATG7, ATG5, BECLIN 1 (BECN1), LC3, Sequestrosome1/p62 (SQSTM1/ p62) and the lysosomal membrane protein LAMP2, were evaluated by immunoblot. The lesion volume was measured in cresyl violet-stained brain sections. Autophagy is activated early after NMDA injection but autophagic degradation is impaired due to the cleavage of LAMP2. Twenty-four h after NMDA intrastriatal injection, the autophagic flux is re-established, but LAMP2 cleavage is still observed. The administration of D-BHB through the i.v+pump protocol reduced the content of autophagic proteins and the cleavage of LAMP2, suggesting decreased autophagosome formation and lysosomal membrane preservation, improving autophagic degradation. D-BHB also reduced brain injury. The i.v+i.p administration protocol and the infusion of D-BHB alone showed no effect on autophagy activation or degradation.

Cytoarchitectonics and Neuronal Organization of the Basolateral Nucleus of the Amygdaloid Body of the Brain in Rats Preferring and Not Preferring Alcohol

Analysis of the cytoarchitectonics of the basolateral (BL) nucleus of the amygdaloid body (AB) was performed using cresyl violet-stained frontal paraffin sections of the brains of 10 alcohol-preferring (AP) and 10 alcohol non-preferring (NAP) rats (with equal numbers of males and females in each group). Large and small neurons were found in the BL nucleus. Most large neurons in AP rats were chromoneutral or moderately chromophilic, while those in NAP rats were moderately chromophobic. Studies using the Golgi method showed that long-axon densely branched pyramid-shaped neurons were equivalents of large neurons, while short-axon neurons corresponded to small neurons. Assessment of the ratio of large to small neurons showed that the proportion of the latter in AP rats was 12.3 ± 0.6%, while that in NAP rats was significantly greater, at 19.70 ± 0.23%. These results explain previously reported data showing that the greater specific area of the BL nucleus of the AB in NAP rats was due to the presence of larger numbers of interneurons than present in AP rats.

Abstract WMP78: Prostaglandin D 2 DP1 Receptor Attenuates the Effects of Post-stroke Sleep Disturbance on Stroke Outcomes

Background: Post-stroke sleep disturbance is reported to be an indicator of poor stroke outcomes. The strategic location of the PGD 2 DP1 receptor in specific brain regions regulating cerebrospinal fluid and sleep led us to hypothesize that the DP1 receptor may attenuate the deleterious effects associated with post-stroke sleep disturbance. Methods: First, to test the effect of sleep deprivation on locomotor activity, wildtype mice were individually housed and their activity was monitored 20h/d for 3d. Day zero was used to obtain baseline, while for the next two days, mice were subjected to sleep disturbance for 10h during the sleep phase followed by 10h of no sleep. Second, to test whether a selective DP1 receptor agonist decreases locomotor activity, mice were given an intraperitoneal injection of the agonist and locomotor activity was monitored. Third, to test the effect of post-stroke sleep disturbance on stroke outcomes, wildtype and DP1 -/- mice were subjected to 45min of MCAO and 7d of reperfusion. After MCAO, mice were subjected to post-stroke sleep disturbance for 12h/d for 3d during the mouse sleep phase. Results: Sleep disturbance resulted in higher activity (awake like) during the sleep phase and lower activity during the awake phase. The distance travelled during the awake phase after sleep deprivation was significantly lower (P<0.001). These data suggest that sleep deprivation attenuated locomotor activity. The total ambulatory distance covered by DP1 agonist-treated group 4h post-treatment was 2161.2±846.4cm compared with the vehicle group activity of 7093.5±1953cm (P<0.01), suggesting that the selective pharmacological agent induced sleep. Analysis of the Cresyl violet-stained sections revealed a significantly larger (P<0.05) infarction volume and neurologic deficit in post-stroke, sleep-disturbed DP1 -/- mice compared to similarly treated WT mice. Conclusion: These data show that the PGD 2 DP1 receptor plays a vital role in attenuating the deleterious effects of sleep disturbance on stroke outcomes. Additional studies are underway to further investigate mechanisms and optimal therapeutic conditions. [This work was supported by UF-CTSI (to A.S.A.) and R01NS046400, R01AT007429 (to S.D.)]

THE CYTOARCHITECTONICS AND NEURONAL ORGANIZATION OF THE BASOLATERAL NUCLEUS OF THE AMYGDALA OF THE BRAIN IN ALCOHOL-PREFERRING AND ALCOHOL-NONPREFERRING RATS

Analysis of cytoarchitectonics of the basolateral nucleus (BLN) of the brain amygdala was performed in cresyl violet-stained frontal paraffin sections of the brain in 10 alcohol-preferring (AP) and 10 alcohol-nonpreferring (ANP) rats (with an equal number of male and female animals in each group). The presence of large and small neurons was detected in BLN. Most of the large neurons in AP rats had the character of chromoneutral and moderately chromophilic cells, while in ANP rats these cells were moderately chromophobic. Application of Golgi method demonstrated that the equivalents of large neurons were long-axonal densely branched pyramid-like neurons, and those of small-sized neurons - short-axonal neurons. The determination of the ratio of large and small-sized neurons showed that in AP rats the proportion of latter was 12.3±0.6%, while in the ANP rats it was significantly greater - 19.70±0.23%. These results help to explain the previously obtained data on larger specific area of BLN in amygdala of ANP rats by the presence of greater number of interneurons than in AP rats.

Potential protective effect of honey against chronic cerebral hypoperfusion-induced neurodegeneration in rats

IntroductionTo investigate the neuroprotective potential of Malaysian Tualang honey in chronic cerebral hypoperfusion induced by permanent bilateral common carotid arteries ligation (2VO) in rats. MethodsRats were randomly divided into three groups (n = 10); sham control, honey-untreated 2VO group “2VO” and honey treated 2VO group “2VO + H”. At 10th week of 2VO surgery, all the rat were sacrificed, brains were dissected out, the right hemisphere was processed for histological study, neuronal counts were performed on cresyl violet-stained sections, the number of viable neurons in CA-1 region of the hippocampus were analysed and counted. ResultsThere were damaged, distorted, irregular cells with shrunken cytoplasm and dark pykonotic nuclei in “2VO” rats. Treatment of rats with honey restored the hippocampal cells to their normal structure and reduced loss of neurons in “2VO + H” rats as compared with “2VO” rats. DiscussionThis study shows that Malaysian Tualang honey might have therapeutic potential for the treatment of chronic cerebral hypoperfusion related neurodegenerative disorders.

Effect of prenatal loud music and noise on total number of neurons and glia, neuronal nuclear area and volume of chick brainstem auditory nuclei, field L and hippocampus: A stereological investigation

The present study explores whether prenatal patterned and unpatterned sound of high sound pressure level (110dB) has any differential effect on the morphology of brainstem auditory nuclei, field L (auditory cortex analog) and hippocampus in chicks (Gallus domesticus). The total number of neurons and glia, mean neuronal nuclear area and total volume of the brainstem auditory nuclei, field L and hippocampus of post-hatch day 1 chicks were determined in serial, cresyl violet-stained sections, using stereology software. All regions studied showed a significantly increased total volume with increase in total neuron number and mean neuronal nuclear area in the patterned music stimulated group as compared to control. Contrastingly the unpatterned noise stimulated group showed an attenuated volume with reduction in the total neuron number. The mean neuronal nuclear area was significantly reduced in the auditory nuclei and hippocampus but increased in the field L. Glial cell number was significantly increased in both experimental groups, being highest in the noise group. The brainstem auditory nuclei and field L showed an increase in glia to neuron ratio in the experimental groups as compared to control. In the hippocampus the ratio remained unaltered between control and music groups, but was higher in the noise group. It is thus evident that though the sound pressure level in both experimental groups was the same there were differential changes in the morphological parameters of the brain regions studied, indicating that the characteristics of the sound had a role in mediating these effects.

Abstract 37: Inducible Nitric Oxide Synthase In Infiltrating Neutrophils Mediates Neurotoxicity After Ischemic Brain Injury In Mice

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes to brain damage after cerebral ischemia and iNOS-deficient mice have smaller infarcts after middle cerebral artery occlusion (MCAO) (J Neurosci, 17:9157, 1997). However, the cell type(s) expressing iNOS and mediating tissue damage remain to be defined. We tested the hypothesis that iNOS expressed in neutrophils infiltrating the ischemic brain contributes to tissue injury. To this end, we examined the outcome of cerebral ischemia in iNOS -/- mice reconstituted with iNOS+/+ leukocytes. Lethally irradiated iNOS -/- mice were transplanted with iNOS+/+ bone marrow (BM) to produce chimeric mice expressing iNOS in peripheral leukocytes. Control chimeras were also obtained by transplanting iNOS+/+ or -/- BM in iNOS+/+ or -/- mice, respectively. Five weeks later, mice underwent transient MCAO (n=10/group) and injury volume was assessed after 3 days in cresyl violet-stained brain sections. Chimerism was 89%, indicating successful BM engraftment. iNOS -/- mice transplanted with iNOS+/+ BM had larger infarcts (40±3 mm3; Mean±SE) than iNOS -/- mice receiving iNOS -/- BM (25±3 mm3; p<0.05; ANOVA), and not different from those of iNOS+/+ mice transplanted with iNOS+/+ BM (36±5 mm3; p>0.05). Cell sorting demonstrated iNOS expression in neutrophils infiltrating the infarct of iNOS -/- mice transplanted with iNOS+/+ BM. To confirm that iNOS in neutrophils contributes to tissue damage, adoptive transfer experiments were performed. Neutrophils (Ly6G positive) from iNOS+/+ or -/- mice were isolated by negative selection and 5x105 cells were administered i.v. to iNOS+/+ or -/- mice 24h after MCAO (n=10/group). Consistent with the result in chimeric mice, transfer of iNOS+/+ neutrophils into iNOS -/- mice increased the infarct 3 days after MCAO (vehicle: 35±6; iNOS+/+ neutrophils: 47±6 mm3; p>0.05 from iNOS+/+ mice receiving vehicle), but not when iNOS -/- neutrophils were transferred (38±8 mm3; p>0.05 from iNOS -/- mice). The findings identify iNOS in infiltrating leukocytes, particularly neutrophils, as a mediator of neurotoxicity in the post-ischemic brain and establish iNOS in circulating leukocytes as a therapeutic target for ischemic brain injury. Supported by NIH grant NS34179

Neuroanatomical Correlates of Suicide in Psychosis: The Possible Role of von Economo Neurons

Suicide is the most important incident in psychiatric disorders. Psychological pain and empathy to pain involves a neural network that involves the anterior cingulate cortex (ACC) and the anterior insula (AI). At the neuronal level, little is known about how complex emotions such as shame, guilt, self-derogation and social isolation, all of which feature suicidal behavior, are represented in the brain. Based on the observation that the ACC and the AI contain a large spindle-shaped cell type, referred to as von Economo neuron (VEN), which has dramatically increased in density during human evolution, and on growing evidence that VENs play a role in the pathophysiology of various neuropsychiatric disorders, including autism, psychosis and dementia, we examined the density of VENs in the ACC of suicide victims. The density of VENs was determined using cresyl violet-stained sections of the ACC of 39 individuals with psychosis (20 cases with schizophrenia, 19 with bipolar disorder). Nine subjects had died from suicide. Twenty specimen were available from the right, 19 from the left ACC. The density of VENs was significantly greater in the ACC of suicide victims with psychotic disorders compared with psychotic individuals who died from other causes. This effect was restricted to the right ACC. VEN density in the ACC seems to be increased in suicide victims with psychosis. This finding may support the assumption that VEN have a special role in emotion processing and self-evaluation, including negative self-appraisal.

Neuroprotective effects vary across nonsteroidal antiinflammatory drugs in a mouse model of developing excitotoxic brain injury

Glutamate excitotoxicity is among the main cellular mechanisms leading to perinatal insults in human newborns. We used intracerebral injection of the glutamatergic glutamate N-methyl- d-aspartate-receptor agonist ibotenate to produce excitotoxic lesions mimicking the acquired white matter lesions seen in human preterm infants. We evaluated whether nonsteroidal antiinflammatory drugs (NSAIDs) protected against glutamate excitotoxicity. Aspirin (0.01–100 μg/d), indomethacin (0.1–10 μg/d), paracetamol (10–100 μg/d), or NS-398 (12.5 μg/d) was given daily before ibotenate (P1 to P5) or after ibotenate (P5 to P9). Lesion size was measured on Cresyl Violet-stained brain sections collected on P10. None of the drugs tested alone or in combination increased lesion size. Pretreatment with low- or high-dose aspirin and post-treatment with paracetamol or NS-398 protected against white matter lesions, whereas cortical lesions were decreased by pretreatment with low- or high-dose aspirin or post-treatment with NS-398. The corticosteroid betamethasone (0.18 μg/d) was neuroprotective when given before or after ibotenate and this effect was reversed by concomitant aspirin therapy (10 μg/d). In conclusion, perinatal NSAID administration may have beneficial effects on brain injury if appropriately timed.

Gender Differences in Postresuscitative Brain Structural Changes

Objective: to reveal gender differences in brain structural changes after clinical death and to assess the neuroprotective properties of the hormonal agent Gynodian Depot. Materials and methods. The brain neuronal populations were morphometrical-ly studied in adult albino rats of both sexes which had sustained 10-minute cardiac arrest. At minute 30 after resuscitation, oil solution of estradiol with dehydroepiandrosterone was intramuscularly injected into the study group animals in doses of 0.1 and 5 mg/100 g. The comparison group of animals received the equivalent volumes of saline. Gender- and age-matched intact rats served as a control. An image analysis system of cresyl violet-stained paraffin brain sections was used to determine the density and composition of highly ischemia-perfusion-sensitive populations of pyramidal neurons of Layer V of the sensomo-tor cortex, the CA1 and CA4 hippocampal sectors, and Purkinje cells in the lateral cerebellum. Results. It has been established that there are gender differences in brain morphology in health, which are detectable in the postresuscitative period. The site of lesions has been found to be different in resuscitated rats of different gender. At the same time, male brain lesions are more extensive, i.e. these involve to this or that extent all the examined regions: the cerebellum and CA4 hippocamplal sector exhibit neuronal death; the cortex and CA1 hippocampal sector show dystrophic changes in the nerve cells. In the females, neuronal shedding processes were observed in the CA1 hippocampal sector only. Estradiol + dehydroepiandrosterone treatment has been ascertained to prevent nerve cell death only in the males and to fail to affect the density and composition of the neuronal populations under study in the females. Conclusion. The findings suggest that it is important to identify the structural bases of sexual dimorphism in the body’s reaction to ischemic exposure and that it is necessary to take them into account when developing effective therapy and choosing the optimal treatment policy to correct posthypoxic encephalopathies. Key words: gender differences, postresuscitative neuronal changes, hormonal therapy, morphometry.

CD47 gene knockout protects against transient focal cerebral ischemia in mice

CD47 is a cell surface glycoprotein that helps mediate neutrophil transmigration across blood vessels. The present study was performed to determine whether absence of the CD47 gene decreases focal ischemic brain damage. Mice were subjected to 90 min middle cerebral artery occlusion. CD47 knockout mice were compared against matching wildtype mice. CD47 expression was checked by Western blotting. Infarct volume and ischemic brain swelling were quantified with cresyl violet-stained brain sections at 24 and 72 h after ischemia. The tight junction protein claudin-5 was detected by imunohistochemistry. Two surrogate markers of neuroinflammation, brain levels of matrix metalloproteinase-9 (MMP-9) and infiltration of neutrophils, were assessed by immunohistochemistry. Western blots confirmed that CD47 was absent in knockout brains. Ischemia did not appear to upregulate total brain levels of CD47 in WT mice. In CD47 knockout mice, infarct volumes were reduced at 24 and 72 h after ischemia, and hemispheric swelling was decreased at 72 h. Loss of claudin-5 was observed in ischemic WT brain. This effect was ameliorated in CD47 knockout brains. Extravasation of neutrophils into the brain parenchyma was significantly reduced in CD47 knockout mice compared to wildtype mice. MMP-9 appeared to be upregulated in microvessels within ischemic brain. MMP-9 levels were markedly lower in CD47 knockout brains compared to wildtype brains. We conclude that CD47 is broadly involved in neuroinflammation, and this integrin-associated-protein plays a role in promoting MMP-9 upregulaton, neutrophil extravasation, brain swelling and progression of acute ischemic brain injury.

Chronic brain injury and behavioral impairments in a mouse model of term neonatal strokes

Stroke in term neonates remains a significant cause of long-term neurological morbidity. This study was designed to assess the relationships between ischemic stroke induced by permanent unilateral carotid ligation in P12 CD1 mice and the structural and functional outcomes in the young mice as a consequence. After P12 ischemic strokes, mice were behaviorally tested using accelerated rotorod, spontaneous alternation on a T-maze, open-field, and cylinder tests between P33 and P39. Brain injury was scored by histology at P40 with cresyl violet-stained coronal sections and computerized quantification of the ischemic injury. The ligation-injured mice were not different from controls on cylinder testing for asymmetric use of their forelimb, or on rotorod measures. In the spontaneous alternation task, however, injured mice demonstrated significantly lower rates of alternation indicating a deficit in working memory. Open-field testing repeated on two consecutive days revealed that the ligated mice were less active than the controls and that they failed to habituate to the open field environment between sessions indicating a learning deficit. Overall, our results demonstrate that ischemia induced by our neonatal stroke model produces behavioral deficits that are consistent with the brain injury.

Effect of the reperfusion after cerebral ischemia in neonatal rats using MRI monitoring

Cerebral hypoxia–ischemia is an important cause of brain injury in the newborn infant. Our purpose was to study magnetic resonance (MR) imaging changes in P7 rat brains submitted to permanent or reversible ischemia. Ischemia was induced by permanent electro-cauterization of the middle cerebral artery combined with a permanent or a transient (50 min) common carotid artery occlusion. The early events during ischemia and reperfusion were investigated by T2-weighted images (T2WI) at 1 and 3 h and by serial diffusion-weighted images (DWI) during 3 h in a 7 T magnet with a standard weighted diffusion sequence ( b = 1282.04 s mm − 2 ) and a SEMS sequence. Within the first hour after MCA occlusion, the T2WI areas of contrast enhancement increased to a mean volume of 12.9 ± 6.4%, a steady state still detected at 3 h after the ischemic onset (10.5 ± 2.5%). Contrast enhancement in DWI increased as soon as 15 min of ischemia in all animals up to 50 min after CCA occlusion. In permanent ischemia, DWI abnormalities volume then increased more slowly from 50 min to 3 h after CCA occlusion (+ 25%, n = 5). In reversible ischemia, the DWI abnormalities volume either moderately decreased and reached a plateau (− 8.4%, n = 4) or dramatically decreased (− 53.0%, n = 3). Both T2WI and DWI evidenced a “patchy” pattern of recovery as also shown on cresyl violet-stained sections. In contrast to the adult, early ischemic injury in P7 rat brains is detected as an increase in hyper-intensities both in T2WI and DWI. Our data indicate that reperfusion is able to block edema evolution after neonatal stroke and that early T2WI and more accurately DWI allow to distinguish between different patterns of injury in reversible ischemia.

Inhibition of MLK3‐MKK4/7‐JNK1/2 pathway by Akt1 in exogenous estrogen‐induced neuroprotection against transient global cerebral ischemia by a non‐genomic mechanism in male rats

Numerous studies have demonstrated the neuroprotective effects of estrogen in experimental cerebral ischemia. To investigate molecular mechanisms of estrogen neuroprotection in global ischemia, immunoblotting, immunohistochemistry and Nissel-staining analysis were used. Our results showed that chronic pretreatment with beta-estradiol 3-benzoate (E2) enhanced Akt1 activation and reduced the activation of mixed-lineage kinase 3 (MLK3), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase 1/2 (JNK1/2) in the hippocampal CA1 subfield during reperfusion after 15 min of global ischemia. In addition, E2 reduced downstream JNK nuclear and non-nuclear components, c-Jun and Bcl-2 phosphorylation and Fas ligand protein expression induced by ischemia/reperfusion. Administration of phosphoinositide 3-kinase (PI3K) inhibitor LY 294,002 prevented both activation of Akt1 and inhibition of MLK3, MKK4/7 and JNK1/2. The interaction between ERalpha and the p85 subunit of PI3K was also examined. E2 and antiestrogen ICI 182,780 promoted and prevented this interaction, respectively. Furthermore, ICI 182,780 blocked both the activation of Akt1 and the inhibition of MLK3, MKK4/7 and JNK1/2. Photomicrographs of cresyl violet-stained brain sections showed that E2 reduced CA1 neuron loss after 5 days of reperfusion, which was abolished by ICI 182,780 and LY 294,002. Our data indicate that in response to estrogen, ERalpha interacts with PI3K to activate Akt1, which may inhibit the MLK3-MKK4/7-JNK1/2 pathway to protect hippocampal CA1 neurons against global cerebral ischemia in male rats.

The effect of pre- and posttreatment with diazoxide on the early phase of chronic cerebral hypoperfusion in the rat

Diazoxide has been identified as a mitochondrial, ATP-dependent K + channel opener, and a potentially neuroprotective compound under ischemic conditions. We set out to characterize the consequences of various treatment strategies with diazoxide in a rat model of chronic cerebral hypoperfusion. Cerebral hypoperfusion was induced by permanent, bilateral occlusion of the common carotid arteries (2VO, n = 36), sham-operated rats serving as controls (SHAM, n = 29). Diazoxide or its vehicle was administered i.p. daily (5 × 0.5 mg/kg/0.25 ml) or as a bolus injection (5 mg/kg/0.25 ml) before surgery or daily after surgery (5 × 0.5 mg/kg/0.25 ml). Spatial learning performance was assessed 1 week after 2VO in the Morris maze. Hippocampal pyramidal cell loss was assessed on cresyl violet-stained sections, while glial reactivity was labeled immunocytochemically. Daily or bolus pretreatment with diazoxide significantly improved 2VO-related learning impairment, whereas posttreatment was ineffective. The number of CA1 pyramidal neurons was reduced by 2VO, which was prevented by repeated or bolus pretreatment with diazoxide. Astrocyte proliferation and microglial activation were enhanced by posttreatment with diazoxide in the hippocampus CA1 area of 2VO animals as compared with SHAM. These data demonstrate that the neuroprotective effect exerted by diazoxide depends on the time of administration with respect to the onset of ischemia; pretreatment but not posttreatment with the compound has proved to be neuroprotective in chronic cerebral hypoperfusion. Thus, pretreatment with diazoxide offers therapeutical prospects for the treatment of cerebral ischemia.

Inducible Nitric Oxide Synthase Contributes to Gender Differences in Ischemic Brain Injury

Estrogens have antiinflammatory actions and protect the brain from ischemic injury. Cerebral ischemia is accompanied by an inflammatory reaction that contributes to the tissue damage, an effect mediated in part by toxic amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS). Therefore, estrogens may protect the female brain by modulating postischemic iNOS expression. To test this hypothesis, we studied whether iNOS plays a role in the mechanisms of the reduced susceptibility to ischemic injury observed in female mice. The middle cerebral artery was occluded for 20 mins using an intraluminal filament in C57Bl/6 mice, and infarct volume was assessed 3 days later in cresyl violet-stained sections. Infarcts were 53% smaller in female mice than in males (P < 0.05), a reduction abolished by ovariectomy (OVX) and reinstated by estrogen replacement. In normal female mice, postischemic iNOS mRNA was lower than in males (P < 0.05). Ovariectomy increased iNOS mRNA after ischemia and estrogen replacement blocked this effect. Furthermore, the iNOS inhibitor aminoguanidine reduced infarct volume in male, but not in female, mice. Similarly, male iNOS-null mice had smaller infarcts than wild-type mice, but female iNOS nulls were not protected. Ovariectomy and OVX with estrogen replacement did not affect infarct volume in iNOS-null female mice. The findings suggest that the neuroprotection conferred by estrogens is, in part, related to attenuation of iNOS expression. Such attenuation could result from the potent antiinflammatory effects of estrogens that downregulate iNOS expression via transcriptional or posttranscriptional mechanisms.

Genetic Ablation of Polysialic Acid Causes Severe Neurodevelopmental Defects Rescued by Deletion of the Neural Cell Adhesion Molecule

Poly-alpha2,8-sialic acid (polySia) is a unique modification of the neural cell adhesion molecule, NCAM, tightly associated with neural development and plasticity. However, the vital role attributed to this carbohydrate polymer has been challenged by the mild phenotype of mice lacking polySia due to NCAM-deficiency. To dissect polySia and NCAM functions, we generated polySia-negative but NCAM-positive mice by simultaneous deletion of the two polysialyltransferase genes, St8sia-II and St8sia-IV. Beyond features shared with NCAM-null animals, a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death was observed. These drastic defects were selectively rescued by additional deletion of NCAM, demonstrating that they originate from a gain of NCAM functions because of polySia deficiency. The data presented in this study reveal that the essential role of polySia resides in the control and coordination of NCAM interactions during mouse brain development. Moreover, this first demonstration in vivo that a highly specific glycan structure is more important than the glycoconjugate as a whole provides a novel view on the relevance of protein glycosylation for the complex process of building the vertebrate brain.

Hydroxyl radicals generated in the rat spinal cord at the level produced by impact injury induce cell death by necrosis and apoptosis: protection by a metalloporphyrin

We previously measured the time courses of hydrogen peroxide (H 2O 2), hydroxyl radical ( •OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of •OH causes cell death. OH was generated by administering H 2O 2 and Fe 2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of •OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post- •OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that •OH generated in the cord induced significant losses of neurons in both Cresyl Violet ( P<0.001) and anti-NSE-stained sections ( P<0.001), and of astrocytes in GFAP-stained sections ( P=0.001). •OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control ( P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced •OH-induced death of neurons ( P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes ( P=0.03). It also reduced the numbers of TUNEL-positive neurons ( P=0.01). Electron microscopy confirmed that generated •OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated •OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces •OH-induced cell death, perhaps by removing H 2O 2 administered in the tissue, thereby blocking formation of •OH, and also by scavenging downstream reactive species.