Dark Neurons of the Sensorimotor Cortex of White Rats after Acute Incomplete Ischemia in Terms of Artifacts Fixation and Neuroglial Relationships

The aimof research was to study morphofunctional signs of the sensorimotor "dark neurons" formation in the cerebrum of sexually mature white rats under normal conditions and in 40 minutesafter the common carotid arteries (CCA) occlusion.Material and methods.Acute ischemia was simulated in white Wistar rats by a 40-min CCA occlusion. The brain was fixed by immersion and perfusion methods. A comparative morphometric assessment of cyto-and gliocytoarchitectonics of the neocortex was carried out under normal conditions (n = 6), 1 (n = 6), 3 (n= 6), and in 7 days (n = 6) after the common carotid arteries (CCA) occlusion. The Nissl staining, hematoxylin and eosin staining, immunohistochemical typing NSE, MAP-2, HSP-70, p38, CASP3, GFAP and AIF1 were applied. The numerical density of normochromic and hyperchromic (dark) pyramidal neurons, oligodendrocytes, microgliocytes, and the relative area of edema –swelling zones were determined. Statistical hypotheses were tested by nonparametric methods using Statistica 8.0 software.Results.The method of the brain fixation significantly influenced the content of the artifact dark neurons,which are similar in morphology and tinctorial properties to degeneratively altered dark neurons. The appearance of reversibly and irreversibly degeneratively altered dark neurons in the sensorimotor cortex after the CCA occlusion was accompanied by an increase in the relative area of edema –swelling zones (control –5.4%, 1st day –17.6%). The maximum content of degeneratively altered dark neurons (53%) was found in layer V in 7 days after ischemia. According to the findings of immunohistochemical typing NSE, MAP-2, HSP-70, p38, CASP3, the specific proteins of the most degeneratively altered dark neurons were retained, ensuring their restoration and the neural network functioning. The total number density of sensorimotor neurons decreased by 26.4% (p =0.001) in layer III, and by 18.5% in layer V (Mann –Whitney U Test; p = 0.01) in 7 days after the intervention. The revealed changes were of a diffuse focal character. In the zones of degeneratively altered dark neurons accumulation, the content of astrocytes, microgliocytes and oligodendrocytes increased. The neuro-astroglial index (control –1.62) increased to 2.72 in 3 days after the CCA occlusion. The peak in the number density of microgliocyte s was noted in 1 day, and the oligodendrocytes in 7 days after acute incomplete ischemia (Mann –Whitney U Test; p0.001).Conclusion.After a 40-minute common carotid arteries occlusion, dark neurons were detected in layers III and V of the sensorimotor cortex with underlying hydropic dystrophy; their cytomorphological pro perties indicated the dynamics of their in vivo degenerative changes. Restoration of degeneratively altered dark neurons was accompanied by an increase in the number of satellite oligodendrocytes, astrocytes and microgliocytes. The revealed changes were considered as one of the variants of reversible changes in neurons in response to moderate ischemic damage.

Histopathological changes of neuronal tissue following the use of hydrogen peroxide in neurosurgical procedures

Background: Hydrogen peroxide (HP) is routinely used in neurosurgical procedures to achieve surgical hemostasis. However, its safety profile is still debatable with various reports depicting range of adverse effects on neuronal tissue. The objective of this paper is to evaluate the safety and efficacy of HP as a hemostatic agent in normal neuronal tissue during neurosurgical procedures conducted on rats. Methods: One hundred rats were divided into three groups. The first and third group underwent cortical irrigation with HP and the second group underwent spinal irrigation with HP. All groups were irrigated with different concentrations of HP (1%, 3%, or 6%) for 3 min and tissue biopsies were obtained immediately afterwards (Groups A and B) or 1 week after HP irrigation (Group C). Study specimens were examined histologically and compared to control tissue. Results: All rats showed normal behavioral, functional, and motor neurological activity following the procedures. Histopathologically, dark neurons were observed in all HP exposed tissue. The cytoplasm revealed condensed and dark Nissl substance and the neurites and axons exhibited a corkscrew morphology. No ischemic changes or inflammatory infiltrates were detected. The majority of dark neurons were observed at the periphery of tissue fragments. These findings were present and consistent in both the short- and long-term groups. Conclusion: HP irrigation showed no significant short- or long-term clinical and histopathological changes in comparison to normal saline when used on rats’ neuronal tissue. This may confirm the safety of intraoperative HP usage as hemostatic agent during neurosurgical procedures.

Hydropic degeneration of the sensorimotor cortex of white rats in the context of the formation of dark neurons and changes in neuroglial relationships following short-term common carotid arteries occlusion

Introduction. Neurodegenerative processes are key in the development of a number of diseases — strokes, Parkinson’s and Alzheimer’s diseases, epilepsy, and traumatic brain injury. They include primary and secondary structural and functional changes in the nervous tissue, as well as the death of neurons and complete loss of functions. Aim. To study the manifestations of hydropic degeneration and reorganization of glio-cytoarchitectonics during the formation of dark neurons in the sensorimotor cortex (SMC) of the cerebrum of mature white rats in 40 min after the common carotid arteries occlusion (CCAO). Materials and methods. A 40-minute CCAO was simulated in white Wistar rats. The brain was fi xed by perfusion method. Morphometric assessment of manifestations of edema — swelling, cyto- and glio-cyto-architectonics of SMC was performed normally (n = 6, control group), on the 1st (n = 6), 3rd (n = 6) and 7th days (n = 6) after CCAO. The Nissl staining, hematoxylin and eosin, immunohistochemical typing NSE, MAP-2, GFAP and AIF1 were applied. The relative area of edema-swelling zones, the numerical density of normochromic and dark pyramidal neurons, oligodendrocytes (OD), microgliocytes (MG) were determined. Results. The high content (20–50%) of dark neurons after CCAO was accompanied by a 3.3-fold increase in the relative area of edema-swelling zones (r = 0.82, p = 0.01). Specifi c proteins (NSE, MAP-2) of most dark neurons were preserved. The total numerical density of SMC neurons decreased by 26.4% (layer III, p = 0.001) and 18.5% (layer V, p = 0.01) after 7 days of CCAO. The content of astrocytes, MG and OD increased. The peak in the number density of MG was observed in the 1st day, and OD — in the 7th day after acute subtotal ischemia (p ≤ 0.001). The revealed changes were of a diffuse-focal nature. Conclusion. After a 40-minute CCAO, the content of dark neurons in SMC increased and, as a result, signs of hydropic degeneration appeared. Against this background, the number of satellite OD, astrocytes and MG increased. Probably, edema-swelling, active MG and astrocytes previously (on the 1st – 3rd day) sanitate the nerve tissue, ensuring its subsequent (on the 7th day) structural and functional recovery with the participation of OD

Protective Effect of Curcumin on the Density of Hippocampal Dark Neurons in Mice Model of Aging Induced by D-Galactose: A Histopathological Study

Background: Curcumin is the most active ingredient in turmeric root of Curcuma longa of the Zingiberaceae family and has a potent antioxidant activity. This study aimed at investigating the effects of curcumin with various doses on the density of dark neurons in the hippocampus of induced D-galactose aging mice model. This experimental study was conducted on 40 adult male BALB/c mice. Materials and Methods: We randomly divided animals into 5 groups: D-galactose, control, and curcumin 1, curcumin 2, and curcumin 3 groups. D-galactose (300 mg/kg) was intraperitoneally injected into the D-galactose group for 6 weeks. D-galactose and doses of 25, 50, and 100 mg of curcumin were administrated, respectively, to the curcumin groups 1-3 every day for 14 days. After 6 weeks, the mice’s brains were stained with toluidine blue after tissue passage. Then, the mean dark neuron volume density in each unit of the tissue was assessed using stereological formulas. The obtained data were analyzed by Aanlysis of Variance (ANOVA) in SPSS. Results: Compared with the control group, the average number of dark neurons in the hippocampus significantly increased following the administration of D-galactose (P=0.001). The average dark neurons frequency in the hippocampus significantly decreased in the 50 and 100 mg/kg curcumintreated groups (P=0.001 for both) compared to that of the D-galactose treated animals. Conclusion: The results of this study showed that treatment with 100 mg/kg of curcumin reduced the number of dark neurons in the hippocampus of the aging mice. It seems that curcumin decreases dark neurons via the reduction of apoptosis. Also, curcumin is a powerful antioxidant and affects the level of antioxidant status in the brain.

Loss of Immunohistochemical Reactivity in Association With Handling-Induced Dark Neurons in Mouse Brains

The handling-induced dark neuron is a histological artifact observed in brain samples handled before fixation with aldehydes. To explore associations between dark neurons and immunohistochemical alterations in mouse brains, we examined protein products encoded by Cav3 (neuronal perikarya/neurites), Rbbp4 (neuronal nuclei), Gfap (astroglia), and Aif1 (microglia) genes in adjacent tissue sections. Here, dark neurons were incidental findings from our prior project, studying the effects of age and high-fat diet on metabolic homeostasis in male C57BL/6N mice. Available were brains from 4 study groups: middle-aged/control diet, middle-aged/high-fat diet, old/control diet, and old/high-fat diet. Young/control diet mice were used as baseline. The hemibrains were immersion-fixed with paraformaldehyde and paraffin-embedded. In the hippocampal formation, we found negative correlations between dark neuron hyperbasophilia and immunoreactivity for CAV3, RBBP4, and glial fibrillary acidic protein (GFAP) using quantitative image analysis. There was no significant difference in dark neuron hyperbasophilia or immunoreactivity for any protein examined among all groups. In contrast, in the hippocampal fimbria, old age seemed to be associated with higher immunoreactivity for GFAP and allograft inflammatory factor-1. Our findings suggest that loss of immunohistochemical reactivity for CAV3, RBBP4, and GFAP in the hippocampal formation is an artifact associated with the occurrence of dark neurons. The unawareness of dark neurons may lead to misinterpretation of immunohistochemical reactivity alterations.