Whether apoptotic cell death occurs in cerebellar Purkinje cells following transient global ischemia remains unclear. Histologic, immunofluorescent and ultramicroscopic methods were used to assess ischemic outcomes in rats. A pilot study using fluorescence labeling [TUNEL-caspase 3-activated peptide (C3AP)-DAPI] revealed key apoptotic characteristics including nuclear TUNEL-positive, nuclear membrane blebs (NMBs), and cytoplasmic C3AP-positive structures, in addition to transport of TUNEL-positive round structures into cytoplasm and projections in ischemic Purkinje cells but not in controls 4 days after ischemia. A formal follow-up study confirmed that 8% of Purkinje cells 4 days following ischemia exhibited TUNEL-positivity and/or NMBs, while Purkinje cells in controls did not. TUNEL-positive Purkinje cells displayed reduced intensity of Calbindin D28K-ifl/MitoTracker (living cell markers) labeling as compared to controls (p < 0.01). Ultramicroscopic evidence of apoptosis included mitochondrial fragmentation and loss in addition to NMBs and cytosolic deposit of nuclear autophagosomes. Interestingly, 71% of the Purkinje cells exhibited autophagy activity after ischemia. Ultramicroscopic characteristics of survival in ischemic Purkinje cells included a centrally located nucleus, no significant chromatin condensation, stable nuclear and intact cytoplasmic membranes, and normal peripheral spacing of the Purkinje cells. In conclusion, four days after transient global ischemia, cerebellar Purkinje cells exhibited both apoptotic and survival characteristics. Further research warrants investigation of the underlying mechanisms.

Background: A recent report links heightened prenatal amniotic estrogen levels to an increased risk of autism spectrum disorder (ASD). In this study, we examined the developmental effects of perinatal estrogen treatment on stem cell activity in weaned rats. Methods: Sprague-Dawley rats received ethinyl estradiol (EE2, 10 mu g/kg/day) or vehicle orally from gestational day 6 until parturition. Offspring were then treated with the same daily dose from postnatal days (PNDs) 1-21. The effects of perinatal estrogen treatment on stem cell activities in the subgranular zone (SGZ) of the hippo campus and the piriform cortex were evaluated in male and female rat pups. Results: EE2 treatment increased the total Ki67-immunoreactive (Ki67-ir) cell counts in the SGZ of males and females (p < 0.05). However, no treatment or sex differences were detectable in the density of the doublecortin (DCX)-immunoreactive (DCX-ir) deposits in the hippocampus. In the piriform cortex, no treatment or sex differences were detected in Ki67-ir cell counts. However, the EE2 treatment significantly reduced the DCX-ir cell count in male, but not female rats (male EE2 group = 292 +/- 22/mm(2), male vehicle group = 402 +/- 19/mm(2), female EE2 group = 342 +/- 15/mm(2), female vehicle group = 331 +/- 9/mm(2)). Conclusions: Perinatal estrogen treatment increased hippocampal Ki67-ir cell counts in both sexes and selectively reduced DCX-ir cell counts in the piriform cortex of males. These data suggest that exposure to abnormally high levels of estrogens early in life may have an impact on neural cell development. Alterations in development so early in life may have long-term cognitive impact.

A pericyte-centered theory suggesting that embolisms occurring within the microvasculature of a neurovascular unit that can result in either parenchymal hemorrhage or intravascular congestion is presented here. Dysfunctional microvascular pericytes are characterized by their location in the neurovascular unit, either on the arteriole or venule side. Pathophysiological and pathological changes caused by coronavirus disease 2019 (COVID-19) include pulmonary hypertension, edema, focal hemorrhage, microvascular congestion, and thrombosis. In this paper, the application of the pericytes-centered hypothesis to COVID-19 has been presented by proposing the concept of a pulmonary neurovascular unit (pNVU). The application of this concept implies that human lungs contain approximately 300 million pNVUs. This concept of existing local regulation of microvascular blood flow is supported by the observation of pathophysiology in pulmonary embolism and in acute high-altitude illness. The autonomic control seen in these three disease states matches blood flow with oxygen supply in each pNVU to maintain physiological blood oxygen saturation level. This paper illustrates how the malfunction of microvascular pericytes may cause focal hemorrhage, edema or microvascular congestion and thrombosis. A bypass existing in each pNVU would autonomically deviate blood flow from a COVID-19-affected pNVU to other healthy pNVUs. This action would prevent systemically applied medicines from reaching the therapeutic threshold in COVID-19-affected pNVUs. While testing this hypothesis with experimental evidence is urgently needed, supporting therapy aimed at improving microcirculation or rebuilding the physiological function of microvascular pericytes is recommended as a potentially effective treatment of COVID-19.

A pericyte-centered hypothesis suggests that embolisms occurring within microvasculature of a neurovascular unit can result in either parenchymal hemorrhage or intravascular congestion. Dysfunctional microvascular pericytes are featured depending on their location in the neurovascular unit. I extend the hypothesis by proposing a concept of pulmonary neurovascular unit (pNVU). In simulating pathophysiology in pulmonary embolisms, acute high-altitude illness and COVID-19, an existing local regulation of microvascular blood flow is believable. This control balances blood flow with oxygen supply to maintain physiological blood oxygen saturation level. We have reported a working module for the neurovascular unit in the sexually dimorphic nucleus of the preoptic area. Pericytes, labeled with alpha-smooth muscle actin immunoreactivity, are significantly denser within the microvasculature of the neurovascular unit in males, signifying their biological functions or potential pathophysiological role in diseases. Noticeably, an illustration provides an explanation of how malfunction of microvascular pericytes causes pulmonary focal hemorrhage, edema or microvascular congestion and thrombi [Fig. 1]. A bypass existing in the pNVU would autonomically deviate blood flow from COVID-19-affected pNVU to other healthy pNVU. Consequentially, systematically applied medicines including chloroquine and/or hydroxychloroquine became valueless due to low concentration of the medicine in the COVID-19-affected regions. Alternatively, a preventive, early antiviral therapy may be efficacious because dysfunctional blood-air exchange precedents and malfunction of pulmonary microcirculation follows. While testing the hypothesis with experimental evidence is urgently needed, supporting therapy aimed at improvement of microcirculation or rebuilding of microvascular pericytes’ physiological function may be recommended during the COVID pandemic.

Background: The pathogenic mechanisms involved in a disastrous scenario, following epidural steroid injections (ESI), remain unclarified. Intra-arterial injection of steroids with needle-penetrating vascular injury would be the culprit, as particulate medicine elicits a brain or spinal cord stroke-like attack. Methods: On the other hand, the limited experimental approaches simulating an accidental steroid intra-arterial injection for ESI conflicted in their results: hemorrhage vs. ischemia. Results: This article dissects the potential pathogenic mechanisms at a neurovascular unit. Noticeably, a schematic representation provides an explanation of how emboli formed by particulate steroids elicit either hemorrhagic, or ischemic lesion. Conclusion: In addition, the development of a rat model with intravertebral artery steroid injection is a proposal to address the unmet need in evaluating steroids and vascular injury in ESI.

Background: The present study aimed at determining pericytes, a missing component in the previously proposed living neurovascular unit (NVU) of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in rats. Materials and Methods: Calbindin D28K-immunoreactivities (CB28-irs) were used to delineate the SDN-POA in which CD13-immunoreactivities (CD13-irs) or alpha-smooth muscle actin-immunoreactivities (alpha SMA-irs), two pericyte biomarkers serving the indexes of pericytes, were tagged using two adjacent brain sections (90-micron intervals). In addition, the nestin-immunoreactive (nestin-ir) cells in the SDN-POA were counted as pericytes referring to additional standards: location and nucleic and cellular morphology. Male SDN-POA volume (5.0 +/- 0.3x10(-3) mm(3)) was significantly larger than the female (1.7 +/- 0.3x10(-3) mm(3)). Within the SDN-POA, the CD13-irs were characterized as dots, densely packed and net-like in distribution, while the alpha SMA-irs, excluding pipe-like or circular structures, appeared as short rod-like structures that were sparsely distributed. Results: The immunoreactive counts of alpha-smooth muscle actin were 353 +/- 57/mm(2) in males and 124 +/- 46/mm(2) in females (p<0.05). On the other hand, densities of the dot-like CD13-irs were similar between males (4009 +/- 301/mm(2)) and females (4018 +/- 414/ mm(2)). There was no difference between the male and the female in the nestin-ir pericyte count in the SDN-POA. Conclusion: In conclusion, the present study adds new information concerning pericytes to the living NVU of the SDN-POA. There is a difference of sex in the count of the alpha SMA-irs in the living NVU of the SDN-POA. However, why such a difference exists warrants further investigations.

Sampling and analysis of cerebrospinal fluid (CSF) is a common clinical practice used in the diagnosis, treatment, and prevention of neurological diseases. A similar interest is the sampling of CSF from rats to bridge the gap between bench-to-bedside work and to foster the development of new CSF biomarkers for clinical use. Here, we describe an improved procedure with an instrument designed in-house, by which rat CSF was successfully collected with indiscernible blood contamination (via the naked eye/surgical microscope amplification). The sampled CSF amounts were over 100 mu l regardless of the animal's body weight, hydration status, and symptoms of systemic damage including, but not limited to, seizure, delusion (such as repeated hemorrhagic self-biting), hematuria, and gastrointestinal bleeding. In adult Sprague-Dawley rats above 300 g, the sampled CSF amounts were reliably at 200 mu l or above with this method. There were no deaths related to the CSF sampling procedure. In conclusion, the present method provides a reliable and reproducible approach for collecting 200 mu l CSF in rats without blood contamination.

The neurovascular unit (NVU) can be conceptualized as a functional entity consisting of neurons, astrocytes, pericytes, and endothelial and smooth muscle cells that operate in concert to affect blood flow to a very circumscribed area. Although we are currently in a “golden era” of bioengineering, there are, as yet, no living NVUs-on-a-chip modules available and the development of a neural chip that would mimic NVUs is a seemingly lofty goal. The sexually dimorphic nucleus of the preoptic area (SDN-POA) is a tiny brain structure (between 0.001~0.007 mm3 in rats) with an assessable biological function (i.e., male sexual behavior). The present effort was undertaken to determine whether there are identifiable NVUs in the SDN-POA by assessing its vasculature relative to its known neural components. First, a thorough and systematic review of thousands of histologic and immunofluorescent images from 201 weanling and adult rats was undertaken to define the characteristics of the vessels supplying the SDN-POA: its primary supply artery/arteriole and capillaries are physically inseparable from their neural elements. A subsequent immunofluorescent study targeting α-smooth muscle actin confirmed the identity of an artery/arteriole supplying the SDN-POA. In reality, the predominant components of the SDN-POA are calbindin D28k-positive neurons that are comingled with tyrosine hydroxylase-positive projections. Finally, a schematic of an SDN-POA NVU is proposed as a working model of the basic building block of the CNS. Such modules could serve the study of neurovascular mechanisms and potentially inform the development of next generation bioengineered neural transplants, i.e., the construct of an NVU neural chip.

Neurites as unique structures have been intensively and extensively studied in efforts to bridge the knowledge gap between molecular signaling pathways and cell morphology, biology, physiology, and pathophysiology. This chapter builds upon a similar chapter in the first edition by addressing molecular mechanisms and genetic modulations underlying neurite development. Cell death mechanisms and stem cell applications with respect to aspects of neurite growth are highlighted along with some of the state-of-the-art approaches to studying neurite development including high-content imaging, three-dimensional (3D) modeling, nanotechnology, and bioengineering. Applications in neurotoxicology are provided for convenient reference.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Neuroendocrine tumors may develop from neuroendocrine cells (NEC), and it has been proposed that pheochromocytoma, a rare catecholamine-producing neuroendocrine tumor, is derived from the chromaffin NECs in the adrenal medulla. In general, the conventional concept of a neuroendocrine system combines aspects of the nervous system with aspects of the endocrine system, highlighting the integration and cooperation between the two. Pulmonary NECs are regarded to be components of the pulmonary neuroendocrine system that consist of a specific group of airway epithelial cells. Chromogranin A, a neuroendocrine cell marker, is present in early human fetuses when the number of chromogranin A-immunoreactive cells surpasses that of insulin- and glucagon-containing cells. In humans, differentiation diagnostic tests can separate normal NECs from cancerous cells because prostatic neuroendocrine tumor cells may also express all of the aforementioned proteins NEC markers. The hypothalamus and the pituitary gland are the major centers of neuroendocrine integration in the body.

Neural stem cell activity at least partially accounts for the postweaning development of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and estrogen selectively mobilizes neural stem cells in the 3rd ventricle stem cell niche (3VSCN). Here, we examined the expression of estrogen receptor β (ERβ) in the SDN-POA and the 3VSCN. A subset of cells within the SDN-POA--delineated with or without calbindin D28K (CB28)-immunoreactivity (ir)--exhibited ERβ-ir. The ependymal cells that expressed nestin within the 3VSCN also expressed ERβ. Interestingly, a few proliferating (Ki67 positive) cells within the 3VSCN and the hypothalamic parenchyma, including the SDN-POA, displayed ERβ-ir. In parallel, a subset of cells in the subventricular zone was double-labeled with nestin and ERβ or Ki67 and ERβ while the subgranular zone exhibited few such double-labeled cells. ERβ is expressed in hypothalamic stem cells that may regulate cell regenerative cycles.

Epidural steroid injections (ESIs) as minimally invasive procedures have been widely used for the relief of neck, arm, back, and leg pain potentially due to spinal stenosis, spondylolysis, or disc herniation. In rare instances, ESI therapy may cause clinical complications, some of which can be catastrophic. The surgical procedure itself including needle penetration, the potential use of contrast media, the injected medications (i.e., conventional steroids), or a combination of these in association with the original cause of the targeted pain which may include local inflammation, may account for such adverse complications. Nevertheless, there is increasing evidence indicating that ischemia and/or hemorrhage (stroke) in the brain and/or spinal cord, following accidental intra arterial injection of the medication is a primary contributor to the severe neurologic events. Descriptions of experimental intra arterial injections simulating the noted catastrophic outcomes associated with ESI therapy are very limited in the literature. Identifying and describing the cause of severe ESI complications will likely rely on the establishment of new experimental models simulating intra vertebral artery or intra-radiculomedullary artery steroid injections.

Exposure to Bisphenol A (BPA) may interfere with brain sexual differentiation. Altered numbers of tyrosine hydroxylase (TH) cells in the rodent anteroventral periventricular nucleus (AVPV) after developmental BPA treatment have been reported; however, definitive conclusions are lacking. The current study incorporated many of the guidelines suggested for endocrine disrupter research. Specifically, ethinyl estradiol (EE2) served as a reference estrogen, exogenous environmental estrogen exposure was controlled, BPA was administered orally, and subjects consumed a low phytoestrogen diet. Here, on gestational days 6–21, Sprague-Dawley rats (10–15/group) were gavaged with 2.5 or 25.0µg BPA/kg/day or 5.0 or 10.0µg EE2/kg/day or the vehicle (5ml of 0.3% aqueous carboxymethylcellulose/kg/day). A naïve control group was weighed and restrained, but not gavaged. Beginning on postnatal day (PND) 1 and continuing until PND 21, the 4 pups/sex/litter were orally treated with the same dose their dam had received. On PND 21, 1/sex/litter was perfused and the brain removed. TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. ANOVA results indicated significantly higher TH-ir cells/mm2 in females (main effect of sex: p<0.01); however, there was no significant effect of treatment or a significant interaction of treatment with sex. In a separate untreated group of PND 21 Sprague-Dawley pups, AVPV volume was quantified and no significant sexual dimorphism was apparent. Similar to our reported results of behavioral assessments, the BPA treatment paradigm used here (2.5 or 25.0µg BPA/kg/day administered orally) does not appear to cause significant alterations in AVPV TH-ir.

The neuroprotective properties of stem cells have been described for various pathophysiological states. Here, we determined the effects of exogenous perinatal estrogen treatment on endogenous neural stem cell activity in the third ventricle stem cell niche (3VSCN) and the caudal third ventricle (C3V). Pregnant Sprague–Dawley rats were gavaged with ethinyl estradiol (EE2, 10 μg/kg/day) or vehicle on gestational days 6–21, and their offspring were similarly treated from birth to weaning on postnatal day 21. At weaning, neural stem cell activity was investigated using the stem cell markers nestin, Ki-67, phosphohistone H3 (PHH3), and doublecortin (DCX). The 3VSCN was characterized by nestin labeling, but little DCX labeling, while both the subventricular (SVZ) and subgranular zones (SGZ) displayed robust DCX expression. Ki-67 cell counts in the 3VSCN were 2.2 to 6.4 times those of the C3V. In the 3VSCN, EE2 treatment significantly increased Ki-67, PHH3, and co-labeled cell counts by 135–207 %, effects which appeared stronger in females. EE2 treatment had only marginally significant effects in the C3V, mildly increasing PHH3 and co-labeled cell counts. Perinatal estrogen treatment selectively increased and mobilized proliferative cells in the 3VSCN at weaning, potentially providing increased neuroprotection. Because PHH3 cells are thought to be in the mitotic phase of the cell cycle and Ki-67 cells can be found in most phases of the cycle, the effect of estrogen treatment on 3VSCN cells appears to involve enhancement of mitosis.

In a clinical study conducted a decade ago, it was proposed that different versions of the gene encoding phosphodiesterase 4D, cAMP-specific ( pde4d) confer different risks for ischemic stroke. Today, however, there continues to be unresolved global discussion of this issue. This short review summarizes the results of clinical genomic and basic research studies. In the acute phase following experimentally-induced stroke, the microvascular expression of PDE4D is increased in association with increased bloodbrain-barrier (BBB) permeability and neuronal death. Treatment with PDE4 inhibitors ameliorates BBB dysfunction and reduces cerebral ischemic damage. Accordingly, experimental approaches using Pde4d knock-out animals for addressing the role of PDE4D in cerebral ischemia are logical next steps. The results of such studies may determine whether or not enhanced PDE4D expression after cerebral ischemia exacerbates stroke outcome and, thus, may provide at least partial resolution to this issue. More importantly, such an approach may provide enhanced opportunities in the search for stroke therapies.

In a clinical study conducted a decade ago, it was proposed that different versions of the gene encoding phosphodiesterase 4D, cAMP-specific (pde4d) confer different risks for ischemic stroke. Today, however, there continues to be unresolved global discussion of this issue. This short review summarizes the results of clinical genomic and basic research studies. In the acute phase following experimentally-induced stroke, the microvascular expression of PDE4D is increased in association with increased bloodbrain-barrier (BBB) permeability and neuronal death. Treatment with PDE4 inhibitors ameliorates BBB dysfunction and reduces cerebral ischemic damage. Accordingly, experimental approaches using Pde4d knock-out animals for addressing the role of PDE4D in cerebral ischemia are logical next steps. The results of such studies may determine whether or not enhanced PDE4D expression after cerebral ischemia exacerbates stroke outcome and, thus, may provide at least partial resolution to this issue. More importantly, such an approach may provide enhanced opportunities in the search for stroke therapies.