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.

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.

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.

We hypothesize that susceptibility to post-traumatic stress disorder（PTSD） may be determined in part by aberrant microtubule-associated protein tau expression in neurons of critical brain structures. The following lines of evidence support this hypothesis. First, epidemiologic data suggest the involvement of genetic factors in the susceptibility to PTSD. Second, the common features of both abnormal tau expression and PTSD include amygdalar and hippocampal atrophy, upregulation of norepinephrine biosynthetic capacity inthe surviving locus coeruleus neurons and dysfunction of N-methyl-D-aspartate-receptors. Finally, our experiments using r Tg4510 mice, a model that over-expresses human mutant tau and develops age-dependent tauopathy, demonstrate that these animals display circling behavior thought to be related to states of anxiety. To detect the potential molecular mechanisms underlying PTSD episodes, laser-assisted/capture microdissection can be used with microarray analysis as an alternative approach to identify changes in gene expression in excitatory and/or inhibitory neurons in critical brain structures（i.e., hippocampus and amygdala） in response to the onset of PTSD.

One of the well-defined sexually dimorphic structures in the brain is the sexually dimorphic nucleus, a cluster of cells located in the preoptic area of the hypothalamus. The rodent sexually dimorphic nucleus of the preoptic area can be delineated histologically using conventional Nissl staining or immunohistochemically using calbindin D28K immunoreactivity. There is increasing use of the calbindin D28K-delineated neural cluster to define the sexually dimorphic nucleus of the preoptic area in rodents. Several mechanisms are proposed to underlie the processes that contribute to the sexual dimorphism (size difference) of the sexually dimorphic nucleus of the preoptic area. Recent evidence indicates that stem cell activity, including proliferation and migration presumably from the 3rd ventricle stem cell niche, may play a critical role in the postnatal development of the sexually dimorphic nucleus of the preoptic area and its distinguishing sexually dimorphic feature: a significantly larger volume in males. Sex hormones and estrogen-like compounds can affect the size of the sexually dimorphic nucleus of the preoptic area. Despite considerable research, it remains unclear whether estrogen-like compounds and/or sex hormones increase size of the sexually dimorphic nucleus of the preoptic area via an increase in stem cell activity originating from the 3rd ventricle stem cell niche.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) has received increased attention due to its apparent sensitivity to estrogen-like compounds found in food and food containers. The mechanisms that regulate SDN-POA volume remain unclear as is the extent of postweaning development of the SDN-POA. Here we demonstrate that the female Sprague-Dawley SDN-POA volume increased from weaning to adulthood, although this increase was not statistically significant as it was in males. The number of cells positive for Ki67, a marker of cell proliferation, in both the SDN-POA and the hypothalamus was significantly higher at weaning than at adulthood in male rats. In contrast, the number of Ki67-positive cells was significantly higher in the hypothalamus but not in the SDN-POA (p>0.05) at weaning than at adulthood in female rats. A subset of the Ki67-positive cells in the SDN-POA displayed the morphology of dividing cells. Nestin-immunoreactivity delineated a potential macroscopic neural stem cell niche in the rostral end of the 3rd ventricle. In conclusion, stem cells may partially account for the sexually dimorphic postweaning development of the SDN-POA.

Background and purpose. While a majority of clinical genomic studies indicate that the gene encoding phosphodiesterase 4D (PDE4D) increases risk of cerebral ischemia, the role of PDE4D in the actual pathogenic mechanisms of ischemia remains unclear. The present study attempts to define a role for PDE4D in the acute phase of cerebral ischemia by monitoring changes in microvascular PDE4D expression and changes in the inside bore of the microvasculature in young and aged rats. Methods. Male F344 rats aged 4- and 24-months were subjected to sham surgery (n=4/group): young control (YC) and aged control (AC) groups or transient global ischemia: young ischemia (YI, n=7) and aged ischemia (AI, n=8) groups. Histological assessments and measurements of PDE4D immunoreactivity in the hippocampus were performed 8 days following ischemia. Results. Perivascular tissue density (defined by an index of the intensity of weak positive per square micron) was significantly lower in the AC group than in the YC group (p<0.05, Fig. 1 E). Transient global ischemia elicited a significant decrease in tissue density in the perimicrovascular space in both young and aged animals as compared to controls ( Fig. 1 E). In addition, internal bore circumference and cross-sectional area increased dramatically as a result of ischemia ( Fig. 1 F). Ischemia also caused a severe loss of hippocampal CA1 neurons ( Fig. 2 A,B,C) in association with significant increases in PDE4D immunoreactivities ( Fig. 2 D-F). Interestingly, hippocampal neuron loss following ischemia paralleled PDE4D expression in microvessels: cell loss was accompanied by much higher levels of perivascular PDE4D expression with this effect being greater in the younger animals. In conclusion, decreases in perivascular tissue density and enlargements in microvascular bore likely indicate an increase in brain-blood barrier (BBB) permeability following the onset of ischemia. This is associated with an increased expression of PDE4D. Increased PDE4D expression following cerebral ischemia may play a role in changing BBB permeability and, thus, secondarily affect ischemic outcome.

Phosphodiesterase 4D (PDE4D) is one of 16 PDEs expressed in cerebral microvessels, and may be involved in regulating blood-brain barrier (BBB) permeability. To assess the possible role of PDE4D in stroke-related injury in young versus aged rats, we measured microvascular PDE4D expression, parenchymal albumin immunoreactivity, and changes in the inside bore of the brain microvasculature. Ischemia caused severe hippocampal CA1 damage, associated with significant increases in vascular PDE4D and parenchymal albumin immunoreactivities. This effect was greater in the younger animals, which also had a greater increase in PDE4D expression. Ischemia significantly decreased tissue density in the perimicrovascular space in both young and aged animals. In addition, internal bore circumference and cross-sectional area of the hippocampal microvessels increased dramatically following ischemia. Increased PDE4D expression following cerebral ischemia may play a role in changing BBB permeability, which could secondarily affect ischemic outcome.

Eleven phosphodiesterase (PDE) families are known, each having several different isoforms and splice variants. Recent evidence indicates that expression of individual PDE family members is tissue-specific. Little is known concerning detailed PDE component expression in brain microvessels where the blood-brain-barrier and the local cerebral blood flow are thought to be regulated by PDEs. The present study attempted to identify PDE family members that are expressed in brain microvessels. Adult male F344 rats were sacrificed and blocks of the cerebral cortex and infratentorial areas were dissected. Microvessels were isolated using a filtration method, and total RNA was extracted. RNA quality and quantity were determined using an Agilent bioanalyzer. The isolated cortical and infratentorial microvessel total RNA amounts were 2720 ± 750 ng (n = 2) and 250 ± 40 ng (n = 2), respectively. Microarrays with 22 000 transcripts demonstrated that there were 16 PDE transcripts in the PDE superfamily, exhibiting quantifiable density in the microvessels. An additional immunofluorescent study verified that PDE4D (cAMP-specific) and PDE5A (cGMP-specific) were colocalized with RECA-1 (an endothelial marker) in the cerebral cortex using both F344 rats and Sprague-Dawley rats (n = 3-6/strain). In addition, PDE4D and PDE5A were found to be colocalized with alpha-smooth muscle actin which delineates cerebral arteries and arterioles as well as pericytes. In conclusion, a filtration method followed by microarray analyses allows PDE components to be identified in brain microvessels, and confirmed that PDE4D and PDE5A are the primary forms expressed in rat brain microvessels.

The present study aimed at identifying early damage index in the cerebellum following total body irradiation (TBI). Adult male CD2F1 mice (n=18) with or without TBI challenge (8.5 Gy irradiation) were assessed for histology and expression of selected immunohistochemical markers including malondiadehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), protein 53 (p53), vascular endothelial growth factor receptor 2 (VEGF-R2), CD45, calbindin D-28k (CB- 28) and vesicular glutamate transport-2 (VGLUT2) in cerebellar folia II to IV. Compared to sham-controls, TBI significantly increased vacuolization of the molecular layer. At high magnification, deformed fiber-like structures were found along with the empty matrix space. Necrotic Purkinje cells were identified on 3.5 days after TBI, but not on 1 day. Purkinje cell count was reduced significantly 3.5 days after TBI. Compared with sham control, overall intensities of MDA and 8-OHdG immunoreactivities were increased dramatically on 1 and 3.5 days after TBI. Expression of VEGF-R2 was identified to be co-localized with 8-OHdG after TBI. This validates microvessel endothelial damage. The p53 immunoreactivities mainly deposited in the granular layer and microvessels after TBI and co-localization of the p53 with the CD45, both which were found within the microvessels. After TBI, CB28 expression decreased whereas the VGLUT2 expression increased significantly; Purkinje cells exhibited a reduced body size and deformity of dendritic arbor, delineated by CB28 immunoreactivity. Substantial damage to the cerebellum can be detectable as early as 1- 3.5 days in adult animals following sublethal TBI. Oxidative stress, inflammatory response and calcium neurotoxicity-associated mechanisms are involved in radiation-induced neuronal damage.