Fluoro-Jade C Research Articles

Inhibition of sodium glucose cotransporters following status epilepticus induced by intrahippocampal pilocarpine affects neurodegeneration process in hippocampus.

Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na(+)/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1μL) or phlorizin, a specific SGLT inhibitor (PZN, 1μL, 50μg/μL), was administered in the hippocampus of rats 30min before PILO (VEH+PILO or PZN+PILO, respectively). The limbic seizures were classified using the Racine's scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C+) were counted 24h and 15days after SE. The PZN-treated rats showed higher (p<0.05) number of WDS when compared with VEH+PILO. There was no difference in seizure severity between PZN+PILO and VEH+PILO groups. However, the pattern of limbic seizures significantly changed in PZN+PILO. Indeed, the class 5 seizures repeated themselves more times (p<0.05) than the other classes in the PZN group at 50min after SE induction. The PZN+PILO animals had a higher (p<0.05) number of FJ-C+ cells in the dentate gyrus (DG), hilus, and CA3 and CA1 of hippocampus, when compared with VEH+PILO. The PZN+PILO animals had a decreased number (p<0.05) of FJ-C+ cells in CA1 compared with VEH+PILO 15days after SE induction. Taken together, our data suggest that SGLT inhibition with PZN increased the severity of limbic seizures during SE and increased neurodegeneration in hippocampus 24h after SE, suggesting that SGLT1 and SGLT2 could participate in the modulation of earlier stages of epileptogenic processes.

BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery after Transplantation in Hypoxic–Ischemic Stroke

Intra-arterial neural stem cell (NSC) therapy has the potential to improve long-term outcomes after stroke. Here we evaluate if pretreatment of NSCs with brain-derived neurotrophic factor (BDNF) prior to transplantation improves cell engraftment and functional recovery following hypoxic-ischemic (HI) stroke. Human embryonic-derived NSCs with or without BDNF pretreatment (1 h, 100 ng/ml) were transplanted 3 days after HI stroke. Functional recovery was assessed using the horizontal ladder test. Cell engraftment was evaluated using bioluminescence imaging (BLI) and histological counts of SC121(+) cells. Fluoro-Jade C (FJC) and NeuN stains were used to evaluate neuroprotection. The effect of BDNF on NSCs was analyzed using a migration assay, immunocytochemistry, Luminex proteomic assay, and RT-qPCR.BLI analysis demonstrated significantly higher photon flux in the BDNF-treated NSC group compared to untreated NSC (p = 0.049) and control groups (p = 0.0021) at 1 week after transplantation. Immunohistochemistry confirmed increased transplanted cell survival in the cortex (p = 0.0126) and hippocampus (p = 0.0098) of animals injected with BDNF-treated NSCs compared to untreated NSCs. Behavioral testing revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC and control groups (p < 0.001) over the 1-month period (p < 0.001) following transplantation. A significant improvement in performance was found in the BDNF-treated NSC group compared to the control group at 14, 21, and 28 (p < 0.05) days after transplantation. The cortex and hippocampus of the BDNF-treated NSC group had significantly more SC121(+) NSCs (p = 0.0125, p = 0.0098), fewer FJC(+) neurons (p = 0.0370, p = 0.0285), and a higher percentage of NeuN(+) expression (p = 0.0354) in the cortex compared to the untreated NSC group. BDNF treatment of NSCs resulted in significantly greater migration to SDF-1, secretion of M-CSF, VEGF, and expression of CXCR4, VCAM-1, Thrombospondins 1 and 2, and BDNF. BDNF pretreatment of NSCs results in higher initial NSC engraftment and survival, increased neuroprotection, and greater functional recovery when compared to untreated NSCs.

MTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy

Background and purposeMammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. MethodsMouse pups (P6) were subjected to hypoxia–ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. ResultsNeuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. ConclusionsmTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP.

N-acetyl-aspartyl-glutamate and inhibition of glutamate carboxypeptidases protects against soman-induced neuropathology

N-acetyl-aspartyl-glutamate (NAAG) is the most abundant neuropeptide in the mammalian brain. In a variety of animal models of brain injury, the administration of NAAG-related compounds, or inhibitors of glutamate carboxypeptidases (GCPs; the enzymes that hydrolyze NAAG), were shown to be neuroprotective. This study determined the impact of the administration of three NAAG-related compounds, NAAG, β-NAAG (a NAAG homologue resistant to degradation), and 2-phosphonomethyl pentanedioic acid (2-PMPA; an inhibitor of GCP enzymes), on the neuropathology that develops following exposure to the nerve agent, soman. When given 1min after soman exposure, NAAG-related drug treatments did not alter the survival rate or body weight loss seen 24h after rats were exposed to soman. Likewise, brain levels of both NAAG and its metabolite, N-acetyl-aspartate (NAA), were substantially decreased 24h after soman, and in particularly vulnerable brain regions the drug treatments were unable to attenuate the reduction in NAA and NAAG levels. Histochemical study indicated there was a dramatic increase in Fluoro-Jade C (FJC) staining, indicative of neuron cell death, 24h after soman exposure. However, in the amygdala and in the entorhinal and piriform limbic cortex, which sustained severe neuropathology following soman intoxication, single or combined injections of NAAG compounds and 2-PMPA significantly reduced the number of FJC-positive cells, and effect size estimates suggest that in some brain regions the treatments were effective. The findings suggest that NAAG neurotransmission in the central nervous system is significantly altered by soman exposure, and that the administration of NAAG-related compounds and 2-PMPA reduces neuron cell death in brain regions that sustain severe damage.

Time course of neuronal death following endothelin-1 induced focal ischemia in rats.

Endothelin-1 (ET-1) induced focal ischemia is increasingly being used as a preclinical model of stroke. Here, we described for the first time, the time course of neuronal death and infarct evolution during the first 7 days following ischemia. We used hematoxylin and eosin (H&E) staining to evaluate infarct progression and Fluoro-Jade C (FJC) to quantify neuronal degeneration at 24, 48, 72h and 7 days after ET-1 injection to the forelimb motor cortex in Sprague-Dawley rats. We found that infarct volume and neuronal degeneration are maximal at 24h post-stroke. Neuronal degeneration is also significantly reduced within 7 days of stroke induction. This study is the first to provide a direct evaluation of both infarct volume evolution and neuronal death time course following ET-1 induced focal ischemia in the forelimb motor cortex. This study describes the short-term time course of neuronal death and brain injury in the ET-1 stroke model, which provides a significant reference when determining the appropriate time to commence neuroprotective or recovery promoting strategies.

Guanosine Protects Against Cortical Focal Ischemia. Involvement of Inflammatory Response.

Stroke is the major cause of death and the most frequent cause of disability in the adult population worldwide. Guanosine plays an important neuroprotective role in several cerebral ischemic models and is involved in the modulation of oxidative responses and glutamatergic parameters. Because the excessive reactive oxygen species produced during an ischemic event can trigger an inflammatory response, the purpose of this study was to evaluate the hypothesis that guanosine is neuroprotective against focal cerebral ischemia, inhibits microglia/macrophages activation, and mediates an inflammatory response ameliorating the neural damage. Permanent focal cerebral ischemia was induced in adult rats, and guanosine was administered immediately, 1, 3, and 6 h after surgery. Twenty-four hours after ischemia, the asymmetry scores were evaluated by the cylinder test; neuronal damage was evaluated by Fluoro-Jade C (FJC) staining and propidium iodide (PI) incorporation; microglia and immune cells were evaluated by anti-Iba-1 antibody; and inflammatory parameters such as interleukins (IL): IL-1, IL-6, IL-10; tumor necrosis factors alpha (TNF-α); and interferon-gamma (INF-γ) were evaluated in the brain tissue and cerebrospinal fluid. The ischemic event increased the levels of Iba-1-positive cells and pro-inflammatory cytokines and decreased IL-10 levels (an anti-inflammatory cytokine) in the lesion periphery. The guanosine treatment attenuated the changes in these inflammatory parameters and also reduced the infarct volume, PI incorporation, and number of FJC-positive cells, improving the functional recovery. Thus, guanosine may have been a promising therapeutic agent for the treatment of ischemic brain injury by reduction of inflammatory process triggered in an ischemic event.

A simple novel technique to induce short‐lasting local brain ischaemia in the rat

Brain ischaemia models are essential to study the pathomechanisms of stroke. Our aim was to investigate the reliability and reproducibility of our novel focal ischaemia-reperfusion model. To induce a cortical transient ischaemic attack, we lifted the distal middle cerebral artery (MCA) with a special hook. The early changes after 2 × 15-min occlusion were observed in the somatosensory evoked responses (SERs). The histological responses to 2 × 15-min MCA occlusion and to 30-, 45- or 60-min ischaemia were examined after a 1-day survival period by 2,3,5-triphenyltetrazolium chloride (TTC) and Fluoro Jade C (FJC) staining. Another group, with 30-min ischaemia, was analysed histologically by FJC, S100 and CD11b labelling after a 5-day survival period. The amplitudes of the SERs decreased immediately at the beginning of the ischaemic period, and remained at a reduced level during the ischaemia. Reperfusion resulted in increasing SER amplitudes, but they never regained the control level. The short-lasting ischaemia did not lead to brain infarction when evaluated with TTC, but intense labelling was found with FJC. The 30-min ischaemia did not result in FJC labelling after 1 day, but marked labelling was observed after 5 days with FJC, S100 and CD11b in the cortical area supplied by the MCA. We present here a novel, readily reproducible method to induce focal brain ischaemia. The ischaemia-reperfusion results in noteworthy changes in the SERs and the appearance of conventional tissue damage markers. This method involves possibilities for precise blood flow regulation, and the setting of the required level of perfusion.

In vitro detection of cytotoxicity using FluoroJade-C

We describe here a novel method for the determination of cytotoxicity in cell cultures using Fluoro-Jade C (FJ-C). FJ-C has been previously used for the assessment of neurodegeneration in fixed brain tissue samples, and has never been utilized in live cell cultures or in different types of cells other than neurons. In the present study we examined the utility of FJ-C for the determination of cytotoxicity in vitro. Various cell cultures were evaluated including neural stem cells, brain microvessel endothelial cells, and SH-SY5Y, PC12 and MDCK cells. Cytotoxicities induced by toxicants in cell cultures, as determined by the FJ-C labeling, were further confirmed by commonly used cytotoxicity assays. This in vitro approach is simple, fast, and sensitive and, thus, has the potential to augment if not replace currently used cell-based cytotoxicity assays.

Brain aging and AD-like pathology in streptozotocin-induced diabetic rats.

Objective. Numerous epidemiological studies have linked diabetes mellitus (DM) with an increased risk of developing Alzheimer's disease (AD). However, whether or not diabetic encephalopathy shows AD-like pathology remains unclear. Research Design and Methods. Forebrain and hippocampal volumes were measured using stereology in serial coronal sections of the brain in streptozotocin- (STZ-) induced rats. Neurodegeneration in the frontal cortex, hypothalamus, and hippocampus was evaluated using Fluoro-Jade C (FJC). Aβ aggregation in the frontal cortex and hippocampus was tested using immunohistochemistry and ELISA. Dendritic spine density in the frontal cortex and hippocampus was measured using Golgi staining, and western blot was conducted to detect the levels of synaptophysin. Cognitive ability was evaluated through the Morris water maze and inhibitory avoidant box. Results. Rats are characterized by insulin deficiency accompanied with polydipsia, polyphagia, polyuria, and weight loss after STZ injection. The number of FJC-positive cells significantly increased in discrete brain regions of the diabetic rats compared with the age-matched control rats. Hippocampal atrophy, Aβ aggregation, and synapse loss were observed in the diabetic rats compared with the control rats. The learning and memory of the diabetic rats decreased compared with those of the age-matched control rats. Conclusions. Our results suggested that aberrant metabolism induced brain aging as characterized by AD-like pathologies.

Pyrrolidine dithiocarbamate (PDTC) inhibits the overexpression of MCP-1 and attenuates microglial activation in the hippocampus of a pilocarpine-induced status epilepticus rat model

The aim of this study was to investigate the effects of pyrrolidine dithiocarbamate (PDTC) on MCP-1 expression and microglial activation in the hippocampus of a rat model of pilocarpine (PILO)-induced status epilepticus (SE). Moreover, seizure susceptibility, frequency and severity as well as brain damage were analyzed and changes in behavior were recorded. Chemokine MCP-1 expression and microglial activation were detected by immunohistochemistry (IHC). Fluoro-Jade C (FJC) and NeuN staining were used for the evaluation of tissue damage. Our results showed that although SE resulted in the upregulation of MCP-1 and microglial activation in the rat hippocampus 24 h after seizure onset, pretreatment with PDTC significantly inhibited the MCP-1 overexpression and attenuated the microglial activation. These effects were accompanied by neurodegenerative amelioration. To the best of our knowledge, these findings indicated for the first time that the activation of the nuclear factor-κB (NF-κB) pathway may contribute to MCP-1 upregulation and microglial activation in the context of epilepsy. PDTC was also shown to exert anticonvulsant activity and to have a neuroprotective effect on the hippocampal CA1 and CA3 regions, potentially through attenuating microglial activation.

Regional Hypothermia Inhibits Spinal Cord Somatosensory-Evoked Potentials without Neural Damage in Uninjured Rats

Both the therapeutic effects of regional hypothermia (RH) and somatosensory-evoked potentials (SSEP) have been intensively studied; however, the in vivo relationship between the two remains unknown. The primary focus of the current study was to investigate the impact of RH on SSEP in uninjured rats, as well as the neural safety of RH on neuronal health. An epidural perfusion model was used to keep local temperature steady by adjusting perfusion speed at 30°C, 26°C, 22°C, and 18°C for 30 min, respectively. Total hypothermic duration lasted up to 3 h. Neural signals were recorded at the end of each hypothermic period, as well as before cooling and after spontaneous rewarming. In addition, the Basso, Beattie, and Bresnahan (BBB) Locomotor Rating Scale was used to evaluate the effects of RH pre- and post-operative, combined with hematoxylin and eosin (H&E) and Fluoro-Jade C (FJC) staining. The results showed a marked declining trend in SSEP amplitude, as well as a significant prolongation in latency only during profound hypothermia (18°C). The BBB scale remained consistent at 21 throughout the entire process, signifying that no motor function injury was caused by RH. In addition, H&E and FJC staining did not show obvious histological injury. These findings firmly support the conclusion that RH, specifically profound RH, inhibits spinal cord SSEP in both amplitude and latency without neural damage in uninjured rats.

Fluoro-Jade C staining in the assessment of neurodegeneration in the thalamus of pilocarpine-treated mice

Objective To detect degenerative neurons by Fluoro⁃Jade C (FJC) that may occur in the thalamus of pilocarpine⁃treated mice and to understand the long⁃term pathologic changes of thalamusand recurrent seizure mechanism in chronic epilepsy. Methods Male adult mice (Kunming stain, n = 10) were divided into pilocarpine ⁃ treated group (n = 5) and controls (n = 5). Pilocarpine ⁃ treated mice were sacrificed at 12 h after status epilepticus. Brain coronal sections in the thalamus level were subject to FJC staining. Finally, FJC⁃stained brain sections were examined under fluorescence microscope to identify the shape and overall distribution of the Fluoro ⁃Jade C positive cells in the thalamus. Results The FJC ⁃ positive staining cells showed a bright green color in the thalamus tissue and fine processes with neuronal profiles. There was a continuity of thalamic damage and neuronal damage in functionally heterogenous thalamic nuclei. They were not detected in control animals. Conclusion This study successfully applied FJC staining to demonstrate massive neurodegeneration in the thalamus, which will benefit further understanding of long ⁃ term brain pathologic changes and recurrent seizure mechanism in temporal lobe epilepsy. DOI：10.3969/j.issn.1672⁃6731.2012.01.014

P.1.g.028 Cognitive effects and Fluoro Jade-C staining in the assessment of 2-vessel occlusion model of brain ischemia in mice

P.1.g.028 Cognitive effects and Fluoro Jade-C staining in the assessment of 2-vessel occlusion model of brain ischemia in mice

Fluoro jade-C staining in the assessment of brain injury after deep hypothermia circulatory arrest

Objective: To evaluate the efficacy of Fluoro Jade-C staining (FJC) in the assessment of brain injury after deep hypothermia circulatory arrest (DHCA). Methods: Six healthy adult miniature male pigs underwent DHCA, the rectal temperature was down to 18°C, circulation was stopped , circulatory arrest was maintained for 60 minutes. On postoperative day 1, perfusion-fixation was performed on brain tissue. Cerebral cortex, hippocampus, cerebellum were taken for sampling. FJC, hematoxylin–eosin staining (HE), nissl staining (NISSL), terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) were performed to detect the histological and pathological changes. Histological scores of all slices were ranked. Comparison between the FJC and other techniques was done by analysis of variance (ANOVA) according to histological scores. Results: All animals survived the operation. On the cerebral cortex, in comparison of FJC between HE, NISSL and TUNEL, the p value was 0.90, 0.40, 0.16 respectively (p>0.05). On the hippocampus, the comparison of FJC with HE, NISSL and TUNEL had a p value of 0.12, 0.23, 0.62 respectively (p>0.05). On the cerebellum, in comparing FJC with HE, NISSL and TUNEL, the p value was 0.96, 0.77, 0.96 respectively (p>0.05). On representative regions, the results of FJC were in accordance with that of TUNEL, NISSL and HE. Furthermore, ascertainment of brain injury is easier with FJC. Conclusion: FJC is a reliable and convenient method to assess brain injury after DHCA.

Effects of postnatal dietary choline manipulation against MK-801 neurotoxicity in pre and postadolescent rats

Prenatal supplementation of rat dams with dietary choline has been shown to provide their offspring with neuroprotection against N-methyl- d-aspartate (NMDA) antagonist-mediated neurotoxicity. This study investigated whether postnatal dietary choline supplementation exposure for 30 and 60 days of rats starting in a pre-puberty age would also induce neuroprotection (without prenatal exposure). Male and female Sprague–Dawley rats (postnatal day 30 of age) were reared for 30 or 60 concurrent days on one of the four dietary levels of choline: 1) fully deficient choline, 2) 1/3 the normal level, 3) the normal level, or 4) seven times the normal level. After diet treatment, the rats received one injection of MK-801 (dizocilpine 3 mg/kg) or saline control. Seventy-two hours later, the rats were anesthetized and transcardially perfused. Their brains were then postfixed for histology with Fluorojade-C (FJ-C) staining. Serial coronal sections were prepared from a rostrocaudal direction from 1.80 to 4.2 mm posterior to the bregma to examine cell degeneration in the retrosplenial and piriform regions. MK-801, but not control saline, produced significant numbers of FJ-C positive neurons, indicating considerable neuronal degeneration. Dietary choline supplementation or deprivation in young animals reared for 30–60 days did not alter NMDA antagonist-induced neurodegeneration in the retrosplenial region. An interesting finding is the absence of the piriform cortex involvement in young male rats and the complete absence of neurotoxicity in both hippocampus regions and DG. However, neurotoxicity in the piriform cortex of immature females treated for 60 days appeared to be suppressed by low levels of dietary choline.

Adenosine A1 Receptor Activation as a Brake on the Microglial Response after Experimental Traumatic Brain Injury in Mice

We reported that adenosine A(1) receptor (A(1)AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A(1)AR KO. We hypothesized that A(1)AR deficiency exacerbates the microglial response and neuronal damage after TBI. A(1)AR KO and WT littermates were subjected to mild controlled cortical impact (3 m/sec; 0.5 mm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24 h or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were stained with Fluoro-Jade C (FJC) at 24 h to evaluate neuronal death in the hippocampus and cresyl violet staining at 7 days to analyze cortical lesion volumes. We also studied the effects of adenosine receptor agonists and antagonists on (3)H-thymidine uptake (proliferation index) by BV-2 cells (immortalized mouse microglial). There was no neuronal death in CA1 or CA3 quantified by FJC. A(1)AR KO mice exhibited enhanced microglial response; specifically, Iba-1 + microglia were increased 20-50% more in A(1)AR KO versus WT in ipsilateral cortex, CA3, and thalamus, and contralateral cortex, CA1, and thalamus (p < 0.05). However, contusion and cortical volumes did not differ between KO and WT. Pharmacological studies in cultured BV-2 cells indicated that A(1)AR activation inhibits microglial proliferation. A(1)AR activation is an endogenous inhibitor of the microglial response to TBI, likely via inhibition of proliferation, and this may represent a therapeutic avenue to modulate microglia after TBI.

Odor enrichment influences neurogenesis in the rostral migratory stream of young rats

The olfactory bulb is one of a few brain structures characterized by high plasticity due to the fact that new neurons are continually integrated into the olfactory bulb circuit throughout life. The new cells originate from the subventricular zone of the forebrain and migrate through the rostral migratory stream (RMS) to the olfactory bulb that also represents the first synaptic relay of the olfactory system. Data accumulating in recent years have confirmed that sensory inputs can influence the level of postnatal neurogenesis in the olfactory bulb. In this study, we studied neurogenesis in the rostral migratory stream of Wistar albino rat pups after exposure to an odor-enriched environment. The rats were olfactory stimulated twice daily with different odorants from the day of their birth up to 1, 2 or 3 weeks, respectively. Using bromodeoxyuridine, a marker of cell proliferation, we found an increased number of proliferating cells in the rostral migratory stream of rat pups submitted to olfactory stimulation. Conversely, the number of dying cells, labeled with the fluorescent dye Fluoro Jade-C, was down-regulated in groups of rats exposed to an odor-enriched environment.

Imipramine enhances cell proliferation and decreases neurodegeneration in the hippocampus after transient global cerebral ischemia in rats

This study was aimed to determine whether imipramine chronic treatment promotes neurogenesis in the dentate gyrus (DG) and interferes with neuronal death in the CA1 subfield of the hippocampus after transient global cerebral ischemia (TGCI) in rats. After TGCI, animals were treated with imipramine (20 mg/kg, i.p.) or saline during 14 days. 5-Bromo-2′-deoxyuridine-5′-monophosphate (BrdU) was injected 24 h after the last imipramine or saline injection to label proliferating cells. In order to confirm the effect of TGCI on neuronal death and cell proliferation, a group of animals was sacrificed 7 days after TGCI. Neurogenesis and neurodegeneration were evaluated by doublecortin (DCX)-immunohistochemistry and Fluoro-Jade C (FJC)-staining, respectively. The rate of cell proliferation increases 7 days but returns to basal levels 14 days after TGCI. There was a significant increase in the number of FJC-positive neurons in the CA1 of animals 7 and 14 days after TGCI. Chronic imipramine treatment increased cell proliferation in the SGZ of DG and reduced the neurodegeneration in the CA1 of the hippocampus 14 days after TGCI. Immunohistochemistry for DCX detected an increased number of newly generated neurons in the hippocampal DG 14 days after TGCI, which was not affected by imipramine treatment. Further studies are needed to evaluate whether imipramine treatment for longer time would be able to promote survival of newly generated neurons as well as to improve functional recovery after TGCI.

Early maternal deprivation in rats induces gender-dependent effects on developing hippocampal and cerebellar cells

Adult animals submitted to a single prolonged episode of maternal deprivation [24h, postnatal day 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. According to the neurodevelopmental theory, these behavioral deficits might be mediated by detrimental neurodevelopmental processes that might be associated, at least partially, with stress-induced corticosterone responses. In order to address this hypothesis, we have focused on the hippocampus and cerebellar cortex, two brain regions that show high density of glucocorticoid receptors, and analyzed possible neuronal and glial alterations by immunohistochemical techniques. To evaluate the presence of degenerated neurons we used Fluoro-Jade-C (FJ-C) staining and for the study of astrocytes we employed glial fibrillary acidic protein (GFAP). Within control animals, females showed significantly more GFAP positive cells than males and a trend towards more FJ-C positive cells. Maternal deprivation induced neuronal degeneration and astroglial changes in the hippocampus and cerebellar cortex of neonatal rats that, in general, were more marked in males. This differential effect may be attributable to a greater vulnerability of males to this kind of early environmental insult and/or to sex-dependent differences in the onset and/or progression of the effects. The present experimental procedure may be instrumental in elucidating sex-dependent mechanisms of neurodevelopmental psychiatric disorders with a basis in early environmental insults.

Application of Fluoro-Jade C in Acute and Chronic Neurodegeneration Models: Utilities and Staining Differences

Recent neuropathological studies have shown that Fluoro-Jade C (FJC), an anionic fluorescent dye, is a good marker of degenerating neurons. However, those studies have mostly examined acute rather than chronic models of neurodegeneration. We therefore compared FJC staining using the intrastriatal 6-hydroxydopamine (6-OHDA)-injected rat as an acute model and the zitter rat as a chronic model, as both show dopaminergic (DA) neurodegeneration. In the 6-OHDA-injected rat, FJC-positive neurons were found in the substantia nigra pars compacta (SNc) before the loss of tyrosine hydroxylase (TH)-positive DA neurons. In the zitter rat, FJC-labeled fibers were first detected at 1 month old (1M) and were considerably increased in the striatum at 4M, whereas FJC-labeled cell bodies were found at 4M, but not at 1M in the SNc. Furthermore, FJC-labeled neurons of the zitter rat showed TH-immunoreactivity in fibers, but little in cell bodies, while those from the 6-OHDA-injected rat showed TH-immunoreactivity even in the cell bodies. These results demonstrate that FJC is a useful tool for detecting chronically degenerating neurons, and suggest that intracellular substances bound to FJC may accumulate in the cell bodies from fibers at a slower rate in the chronic model than in the acute model.