Intracisternal Injection Research Articles

Применение препарата «Мастинол-форте» в терапии клинического мастита у дойных коров

The article presents the results of clinical trials of the veterinary drug «Mastinol-forte». The effectiveness of the drug in the treatment of the serous-catarrhal form of mastitis was studied in the conditions of a dairy farm. The experiment included cows of a black-and-white Holstein breed weighing 500–550 kg, spontaneously ill with a clinical form of mastitis. Five groups of lactating cows of 2–3 lactation were formed, with a yield of 7–8 thousand l/ year, 18 heads each. The therapeutic efficacy of the drug was evaluated clinically by methods, as well as using a dimastin sample and by counting somatic cells in milk samples. It has been established that the use of the drug «Mastinol-forte» as monotherapy for serouscatarrhal clinical mastitis in cows is well tolerated by animals and gives a positive effect both with intramuscular, subcutaneous, and intracisternal injections. However, the best result was obtained with the combined (subcutaneous and intracisternal) injection of mastinol-forte for 5 days at a dose of 7 ml twice a day.

Subacute phase of subarachnoid haemorrhage in female rats: Increased intracranial pressure, vascular changes and impaired sensorimotor function

ObjectiveEarly brain injury (EBI) and delayed cerebral ischemia (DCI) after subarachnoid haemorrhage (SAH) has devastating consequences but therapeutic options and the underlying pathogenesis remain poorly understood despite extensive preclinical and clinical research. One of the drawbacks of most preclinical studies to date is that the mechanisms behind DCI after SAH are studied only in male animals. In this study we therefore established a female rat model of SAH in order to determine subacute pathophysiological changes that may contribute to DCI in females. MethodsExperimental SAH was induced in female rats by intracisternal injection of 300 μL of autologous blood. Sham operation served as a control. Neurological deficits and intracranial pressure measurements were evaluated at both 1 and 2 days after surgery. Additionally, changes in cerebral vascular contractility were evaluated 2 days after surgery using wire myography. ResultsSAH in female rats resulted in sensorimotor deficits and decreased general wellbeing on both day 1 and day 2 after SAH. Intracranial pressure uniformly increased in all rats subjected to SAH on day 1. On day 2 the intracranial pressure had increased further, decreased slightly or remained at the level seen on day 1. Furthermore, female rats subjected to SAH developed cortical brain edema. Cerebral arteries, isolated 2 days after SAH, exhibited increased vascular contractions to endothelin-1 and 5-carboxamidotryptamine. ConclusionIn the subacute phase after SAH in female rats, we observed increased intracranial pressure, decreased wellbeing, sensorimotor deficits, increased vascular contractility and cortical brain edema. Collectively, these pathophysiological changes may contribute to DCI after SAH in females. Previous studies reported similar pathophysiological changes for male rats in the subacute phase after SAH. Thus, prevention of these gender-independent mechanisms may provide the basis for a universal treatment strategy for DCI after SAH. Nevertheless, preclinical studies of potential therapies should employ both male and female SAH models.

Adjuvant Cannabinoid Receptor Type 2 Agonist Modulates the Polarization of Microglia Towards a Non-Inflammatory Phenotype in Experimental Pneumococcal Meningitis.

BackgroundMicroglia initiates and sustains the inflammatory reaction that drives the pathogenesis of pneumococcal meningitis. The expression of the G-protein cannabinoid receptor type 2 (CB2) in the brain is low, but is upregulated in glial cells during infection. Its activation down-regulates pro-inflammatory processes, driving microglia towards an anti-inflammatory phenotype. CB2 agonists are therefore therapeutic candidates in inflammatory conditions like pneumococcal meningitis. We evaluated the effects of JWH-133, a specific CB2 agonist on microglial cells, inflammation, and damage driven by S. pneumoniae in vitro and in experimental pneumococcal meningitis.Materials/methodsPrimary mixed glial cultures were stimulated with live or heat-inactivated S. pneumoniae, or lipopolysaccharide and treated with JWH-133 or vehicle. Nitric oxide and cytokines levels were measured in the supernatant. In vivo, pneumococcal meningitis was induced by intracisternal injection of live S. pneumoniae in 11 days old Wistar rats. Animals were treated with antibiotics (Ceftriaxone, 100 mg/kg, s.c. bid) and JWH-133 (1 mg/kg, i.p. daily) or vehicle (10% Ethanol in saline, 100 µl/25g body weight) at 18 h after infection. Brains were harvested at 24 and 42 h post infection (hpi) for histological assessment of hippocampal apoptosis and cortical damage and determination of cyto/chemokines in tissue homogenates. Microglia were characterized using Iba-1 immunostaining. Inflammation in brain homogenates was determined using membrane-based antibody arrays.Results In vitro, nitric oxide and cytokines levels were significantly lowered by JWH-133 treatment. In vivo, clinical parameters were not affected by the treatment. JWH-133 significantly lowered microglia activation assessed by quantification of cell process length and endpoints per microglia. Animals treated with JWH-133 demonstrated significantly lower parenchymal levels of chemokines (CINC-1, CINC-2α/β, and MIP-3α), TIMP-1, and IL-6 at 24 hpi, and CINC-1, MIP-1α, and IL-1α at 42 hpi. Quantitative analysis of brain damage did not reveal an effect of JWH-133.ConclusionsJWH-133 attenuates microglial activation and downregulates the concentrations of pro-inflammatory mediators in pneumococcal infection in vitro and in vivo. However, we didn’t observe a reduction in cortical or hippocampal injury. This data provides evidence that inhibition of microglia by adjuvant CB2 agonists therapy effectively downmodulates neuroinflammation but does not reduce brain damage in experimental pneumococcal meningitis

Changes in Neuronal Density of the Sensorimotor Cortex and Neurodevelopmental Behaviour in Neonatal Mice with Kaolin-Induced Hydrocephalus

Introduction: Hydrocephalus is a disorder in which the circulation of cerebrospinal fluid is altered in a manner that leads to its accumulation in the ventricles and subarachnoid space. Its impact on the neuronal density and networks in the overlying cerebral cortex in a time-dependent neonatal hydrocephalic process is largely unknown. We hypothesize that hydrocephalus will affect the cytoarchitecture of the cerebral cortical mantle of neonatal hydrocephalic mice, which will in turn modify sensorimotor processing and neurobehaviour. Objective: The purpose of this study is to probe the effect of hydrocephalus on 3 developmental milestones (surface righting reflex, cliff avoidance reflex, and negative geotaxis) and on cortical neuronal densities in neonatal hydrocephalic mice. Methods: Hydrocephalus was induced in 1-day-old mice by intracisternal injection of sterile kaolin suspension. The pups were tested for reflex development and sensorimotor ability using surface righting reflex (PND 5, 7, and 9), cliff avoidance (PND 6), and negative geotaxis (PND 10 and 12) prior to their sacrifice on PND 7, 14, and 21. Neuronal density and cortical thickness in the sensorimotor cortex were evaluated using atlas-based segmentation of the neocortex and boundary definition in 4-μm paraffin-embedded histological sections with hematoxylin and eosin as well as cresyl violet stains. Results: Surface righting and cliff avoidance activities were significantly impaired in hydrocephalic pups but no statistically significant difference was observed in negative geotaxis in both experimental and control pups. The neuronal density of the sensorimotor cortex was significantly higher in hydrocephalic mice than in age-matched controls on PND 14 and 21 (373.20 ± 21.54 × 10⁻⁶ μm² vs. 157.70 ± 21.88 × 10⁻⁶ μm²; 230.0 ± 44.1 × 10⁻⁶ μm² vs. 129.60 ± 3.72 × 10⁻⁶ μm², respectively; p < 0.05). This was accompanied by reduction in the cortical thickness (µm) in the hydrocephalic mice on PND 7 (2,409 ± 43.37 vs. 3,752 ± 65.74, p < 0.05), PND 14 (2,035 ± 322.10 vs. 4,273 ± 67.26, p < 0.05), and PND 21 (1,676 ± 33.90 vs. 4,945 ± 81.79, p < 0.05) compared to controls. Conclusion: In this murine model of neonatal hydrocephalus, the quantitative changes in the cortical neuronal population may play a role in the observed changes in neurobehavioural findings.

Centrally administered orexin prevents lipopolysaccharide and colchicine induced lethality via the vagal cholinergic pathway in a sepsis model in rats

Orexins are neuropeptides implicated in several physiological functions. Accumulating findings suggest a relationship between orexin and sepsis. A recent study demonstrated that orexin acts centrally to improve conditions in sepsis. The present study aims to clarify the precise mechanisms by which central orexin could induce a protective action against septic conditions. We established a new septic model by treating rats with lipopolysaccharide (LPS) and colchicine and used this to examine the effect of brain orexin on survival. Observation of survival was stopped three days after the chemicals injection or at death. We established a lethal model (rats died within 24 h) by injecting subcutaneously a combination of 1 mg/kg LPS and 1 mg/kg colchicine. A Toll-like receptor 4 (TLR4) inhibitor completely blocked lethality, suggesting a vital role of LPS-TLR4 signaling in the process. Intracisternal orexin-A dose-dependently reduced lethality in the sepsis model while neither intracisternal orexin-B nor intraperitoneal orexin-A changed the mortality rate. Vagal stimulation with carbachol or 2-deoxy-D-glucose improved survival and atropine potently blocked the protection by carbachol or 2-deoxy-D-glucose. The orexin-A-induced reduction of lethality was significantly blocked by atropine or surgical vagotomy. Intracisternal injection of an OX1 receptor antagonist blocked the improvement of survival by intracisternal injection of orexin-A, carbachol, or 2-deoxy-D-glucose. These results suggest that orexin acts centrally to reduce the lethality in our septic model treated (LPS and colchicine). Activation of the vagal cholinergic pathway may mediate the action of orexin, and the OX1 receptor in the brain might play a role in the process. Since the efferent vagus nerve mediates anti-inflammatory mechanisms, we speculate that the vagal cholinergic anti-inflammatory pathway is implicated in the mechanisms of septic lethality reduction by brain orexin.

Characterization of Tauopathy in a Rat Model of Post-Stroke Dementia Combining Acute Infarct and Chronic Cerebral Hypoperfusion.

Post-stroke dementia (PSD) is a major neurodegenerative consequence of stroke. Tauopathy has been reported in diverse neurodegenerative diseases. We investigated the cognitive impairment and pathomechanism associated with tauopathy in a rat model of PSD by modeling acute ischemic stroke and underlying chronic cerebral hypoperfusion (CCH). We performed middle cerebral artery occlusion (MCAO) surgery in rats to mimic acute ischemic stroke, followed by bilateral common carotid artery occlusion (BCCAo) surgery to mimic CCH. We performed behavioral tests and focused on the characterization of tauopathy through histology. Parenchymal infiltration of cerebrospinal fluid (CSF) tracers after intracisternal injection was examined to evaluate glymphatic function. In an animal model of PSD, cognitive impairment was aggravated when BCCAo was combined with MCAO. Tauopathy, manifested by tau hyperphosphorylation, was prominent in the peri-infarct area when CCH was combined. Synergistic accentuation of tauopathy was evident in the white matter. Microtubules in the neuronal axon and myelin sheath showed partial colocalization with the hyperphosphorylated tau, whereas oligodendrocytes showed near-complete colocalization. Parenchymal infiltration of CSF tracers was attenuated in the PSD model. Our experimental results suggest a hypothesis that CCH may aggravate cognitive impairment and tau hyperphosphorylation in a rat model of PSD by interfering with tau clearance through the glymphatic system. Therapeutic strategies to improve the clearance of brain metabolic wastes, including tau, may be a promising approach to prevent PSD after stroke.

Receptor for Advanced Glycation End Products (RAGE) Mediates Cognitive Impairment Triggered by Pneumococcal Meningitis.

Pneumococcal meningitis is a life-threatening infection of the central nervous system (CNS), and half of the survivors of meningitis suffer from neurological sequelae. We hypothesized that pneumococcal meningitis causes CNS inflammation via the disruption of the blood-brain barrier (BBB) and by increasing the receptor for advanced glycation end product (RAGE) expression in the brain, which causes glial cell activation, leading to cognitive impairment. To test our hypothesis, 60-day-old Wistar rats were subjected to meningitis by receiving an intracisternal injection of Streptococcus pneumoniae or artificial cerebrospinal fluid as a control group and were treated with a RAGE-specific inhibitor (FPS-ZM1) in saline. The rats also received ceftriaxone 100mg/kg intraperitoneally, bid, and fluid replacements. Experimental pneumococcal meningitis triggered BBB disruption after meningitis induction, and FPS-ZM1 treatment significantly suppressed BBB disruption. Ten days after meningitis induction, surviving animals were free from infection, but they presented increased levels of TNF-α and IL-1β in the prefrontal cortex (PFC); high expression levels of RAGE, amyloid-β (Aβ1-42), and microglial cell activation in the PFC and hippocampus; and memory impairment, as evaluated by the open-field, novel object recognition task and Morris water maze behavioral tasks. Targeted RAGE inhibition was able to reduce cytokine levels, decrease the expression of RAGE and Aβ1-42, inhibit microglial cell activation, and improve cognitive deficits in meningitis survivor rats. The sequence of events generated by pneumococcal meningitis can persist long after recovery, triggering neurocognitive decline; however, RAGE blocker attenuated the development of brain inflammation and cognitive impairment in experimental meningitis.

Pyramidal cell morphology and cell death in the hippocampus of adult mice with experimentally induced hydrocephalus

Background: Hippocampus is a neural structure in the temporal lobe that plays a crucial role in learning and memory. Cognitive impairment with learning disabilities is a common feature in hydrocephalus and is more prominent in adult-onset hydrocephalus. The aim of this study is to describethe morphological alterations in the pyramidal cells of the hippocampus of adult hydrocephalic mice.&#x0D; Method: Hydrocephalus was induced in adult albino mice by intra-cisternal injection of kaolin suspension (250 mg/ml in sterile water). They were sacrificed 7, 14 and 21 days post-induction. Morphological analysis was carried out on hematoxylin and eosinstained coronal sections of the hippocampus: the pyramidal neurons (normal and pyknotic) in the CA1 and CA3 subregions were counted and the pyknotic index (PI) was calculated. The somatic and dendritic features of Golgistained pyramidal neurons were examined by light microscopy in both hydrocephalic and control mice.&#x0D; Result: The PI was significantly greater in the CA1 region of the hippocampus in the hydrocephalic groups compared to the agematched controls. The dendritic processes of pyramidal neurons in the CA1 region were fewer with shorter terminal branches in the hydrocephalic mice than in controls; this was pronounced at 7 days post-induction. In the CA3 region, there was no difference in dendritic arborization between hydrocephalic and control mice.&#x0D; Conclusion: Acute adult-onset hydrocephalus was associated with increased pyknosis and reduced dendritic arborization in hippocampal pyramidal cells in the CA1 but not CA3 region.&#x0D; Keywords: Hippocampal pyramidal cell, Hydrocephalus, Pyknotic index, Golgi stain

Neurobehavioural changes and morphological study of cerebellar purkinje cells in kaolin induced hydrocephalus.

Cerebellar abnormalities are commonly associated with hydrocephalus. However, the effect of hydrocephalus on the otherwise normal cerebellum has been largely neglected. This study assesses the morphological changes in the Purkinje cells in relation to cerebellar dysfunction observed in juvenile hydrocephalic rats. Fifty-five three-week old albino Wistar rats were used, hydrocephalus was induced by intracisternal injection of kaolin (n = 35) and others served as controls (n = 20). Body weight measurements, hanging wire, negative geotaxis, and open field tests were carried out at the onset and then weekly for 4weeks, rats were killed, and their cerebella processed for Hematoxylin and Eosin, Cresyl violet and Golgi staining. Qualitative and quantitative studies were carried out; quantitative data were analyzed using two-way ANOVA and independent T tests at p < 0.05. Hydrocephalic rats weighed less than controls (p = 0.0247) but their cerebellar weights were comparable. The hydrocephalic rats had a consistently shorter latency to fall in the hanging wire test (F(4,112) = 18.63; p < 0.0001), longer latency to turn in the negative geotaxis test (F(4,112) = 22.2; p < 0.0001), and decreased horizontal (F(4,112) = 4.172, p = 0.0035) and vertical movements (F(4,112) = 4.397; p = 0.0024) in the open field test than controls throughout the 4weeks post-induction. Cellular compression in the granular layer, swelling of Purkinje cells with vacuolations, reduced dendritic arborization and increased number of pyknotic Purkinje cells were observed in hydrocephalic rats. Hydrocephalus caused functional and morphological changes in the cerebellar cortex. Purkinje cell loss, a major pathological feature of hydrocephalus, may be responsible for some of the motor deficits observed in this condition.

A functional observational battery for evaluation of neurological outcomes in a rat model of acute bacterial meningitis

BackgroundAcute bacterial meningitis is a disease with a high mortality and a high incidence of neurological sequelae in survivors. There is an acute need to develop new adjuvant therapies. To ensure that new therapies evaluated in animal models are translatable to humans, studies must evaluate clinically relevant and patient-important outcomes, including neurological symptoms and sequelae.MethodsWe developed and tested a functional observational battery to quantify the severity of a variety of relevant neurological and clinical symptoms in a rat model of bacterial meningitis. The functional observational battery included symptoms relating to general clinical signs, gait and posture abnormalities, involuntary motor movements, focal neurological signs, and neuromotor abnormalities which were scored according to severity and summed to obtain a combined clinical and neurological score. To test the functional observational battery, adult Sprague-Dawley rats were infected by intracisternal injection of a clinical isolate of Streptococcus pneumoniae. Rats were evaluated for 6 days following the infection.ResultsPneumococcal meningitis was not lethal in this model; however, it induced severe neurological symptoms. Most common symptoms were hearing loss (75% of infected vs 0% of control rats; p = 0.0003), involuntary motor movements (75% of infected vs 0% of control rats; p = 0.0003), and gait and posture abnormality (67% of infected vs 0% of control rats; p = 0.0013). Infected rats had a higher combined score when determined by the functional observational battery than control rats at all time points (24 h 12.7 ± 4.0 vs 4.0 ± 2.0; 48 h 17.3 ± 7.1 vs 3.4 ± 1.8; 6 days 17.8 ± 7.4 vs 1.7 ± 2.4; p < 0.0001 for all).ConclusionsThe functional observational battery described here detects clinically relevant neurological sequelae of bacterial meningitis and could be a useful tool when testing new therapeutics in rat models of meningitis.

56. TUMOR-HOMING STEM CELL THERAPY INHIBITS THE PROGRESSION OF BREAST CANCER LEPTOMENINGEAL CARCINOMATOSIS

INTRODUCTIONLeptomeningeal carcinomatosis remains one of the most lethal forms of central nervous system metastasis, with a median survival of only 4 months. Effective new therapies are urgently needed to treat this highly aggressive cancer. In this study, we used models of both prophylactic and established leptomeningeal disease to investigate the efficacy of engineered tumor-homing neural stem cells (NSCs) therapy for breast cancer leptomeningeal carcinomatosis.METHODSPersonalized NSC carriers were created using Sox2 overexpression to transdifferentiate human fibroblasts into induced NSCs (iNSCs) that home to cancer cells and carry therapeutic agents to induce tumor kill. Leptomeningeal models were created by engineering MDA-MB231-Br human breast cancer cells with fluorescent and bioluminescent reporters, then using intracisternal injection to inoculate Nude mice with the tumor cells. iNSC therapy was evaluated by infusing iNSCs releasing the pro-apoptotic agent TRAIL into the lateral ventricle of mice either 1 week prior to or 3 days after tumor inoculation for prophylactic or established tumor treatment respectively. Tumor progression in the brain and spinal cord was monitored by serial bioluminescence imaging (BLI).RESULTSSerial BLI showed that intracerebroventricular (ICV) iNSC-TRAIL therapy reduced the volume of metastatic tumor burden 99.49% in the brain and 99.80% in the spine within 2 weeks post-infusion and extended survival from 24 to 42 days. Additionally, prophylactic iNSC-TRAIL therapy delivered ICV markedly delayed tumor development, with tumors in the brain remaining >1000-fold smaller than control through 1-month post-treatment, below the limit of detection in the spinal cord through 1 month, and eliminating mortality through 50 days post-therapy.CONCLUSIONThese data suggest that iNSC therapy could be a promising treatment option for breast cancer patients with leptomeningeal carcinomatosis.

Intracisternal injection of beta-amyloid seeds promotes cerebral amyloid angiopathy

Beta amyloid (Aβ) is a key component of parenchymal Aβ plaques and vascular Aβ fibrils, which lead to cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD). Recent studies have revealed that Aβ contained in the cerebrospinal fluid (CSF) can re-enter into brain through paravascular spaces. However, whether Aβ in CSF may act as a constant source of pathogenic Aβ in AD is still unclear. This study aimed to examine whether Aβ pathology could be worsened when CSF Aβ level was enhanced by intra-cisternal infusion of aged brain extract containing abundant Aβ in TgCRND8 host mice. TgCRND8 mouse is an AD animal model which develops predominant parenchymal Aβ plaques in the brain at as early as 3 months of age. Here, we showed that single intracisternal injection of Aβ seeds into TgCRND8 mice before the presence of Aβ pathology induced robust prion-like propagation of CAA within 90 days. The induced CAA is mainly distributed in the cerebral cortex, hippocampus and thalamus of TgCRND8 mice. Surprisingly, despite the robust increase in CAA levels, the TgCRND8 mice had a marked decrease in parenchymal Aβ plaques and the plaques related neuroinflammation in the brains compared with the control mice. These results amply indicate that Aβ in CSF may act as a source of Aβ contributing to the growth of vascular Aβ deposits in CAA. Our findings provide experimental evidence to unravel the mechanisms of CAA formation and the potential of targeting CSF Aβ for CAA.

Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry.

Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitativedetermination of glymphatic activity, and this will be useful in preclinical studies.

Neuropeptide FF receptor 2 inhibits capsaicin-induced CGRP Upregulation in mouse trigeminal ganglion

BackgroundStimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model.MethodsMice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA.FindingsInfusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice.ConclusionsReducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.

Pericytes in Neurometabolic Diseases

Since brain pericytes guarantee appropriate development and maintenance of the cerebrovascular system, we reviewed their role in neurometabolic diseases (NMDs), a subclass of inborn errors of metabolism that selectively cause neurological sequels and widespread cerebrovascular alterations. Main findings about pericyte involvement in NMDs arise from glutaric acidemia type I (GA-I) models. In this regard, we found that (i) a single intracisternal injection of the main accumulated metabolite (glutaric acid, GA) in rat neonates disturbed the neurovascular unit (NVU) as evidenced by blood-brain barrier hyperpermeability, and altered immunoreactivity of pericyte and astrocyte markers surrounding brain microvessels; (ii) GA-elicited capillary constriction near pericyte somata likely inducing reduced brain blood flow as reported in GA-I patients; (iii) GA-elicited pericyte contraction probably results from a defective interplay among NVU components and could be relevant in case of metabolic decompensation or energetic deficiency. Although pericyte pathological features have been studied in few NMDs, their involvement in NMD pathophysiology is largely unknown. Thus, further studies are needed to identify their roles and therapeutic potentiality.

Electroacupuncture ameliorates corticotrophin-releasing factor-induced jejunal dysmotility in a rat model of stress.

Central injection of corticotrophin-releasing factor (CRF) mimics the effect of stress on gastrointestinal (GI) responses, including inhibition of GI motility. This study was designed to explore the effects of electroacupuncture (EA) on disordered jejunal motility in a rat model of stress induced by intracisternal (IC) injection of CRF. A stress model was established by IC injection of CRF in Sprague-Dawley rats. GI motility was evaluated by assessing gastric emptying (GE), gastrointestinal transit (GIT) and jejunal motility in vivo. EA was performed at ST36. The functional roles of CRF receptor subtype 1 and subtype 2 (CRFr1 and CRFr2) were examined by IC administration of the corresponding selective CRF antagonists. Protein expression of CRFr1 and CRFr2 in the hypothalamus and jejunum was detected by Western blotting. IC injection of CRF significantly inhibited GE, GIT and jejunal motility. EA treatment remarkably improved the disturbed GI motility. Intriguingly, the disordered jejunal motility induced by central CRF was abolished by IC injection of a selective CRFr2 antagonist, indicating the essential role of central CRFr2 in mediating the stress-induced jejunal motor disorder. EA at ST36 decreased central and peripheral expression of CRFr2, which might be one of the potential mechanisms underlying the beneficial effect of EA on jejunal dysmotility in this rat model of stress. This study suggested that EA at ST36 could ameliorate disordered jejunal motility induced by stress, and that this might be associated with the down-regulation of CRFr2.

Ghrelin acts in the brain to block colonic hyperpermeability in response to lipopolysaccharide through the vagus nerve

Brain ghrelin plays a role in gastrointestinal functions. Among them, ghrelin acts centrally to stimulate gastrointestinal motility and induce visceral antinociception. Intestinal barrier function, one of important gastrointestinal functions, is also controlled by the central nervous system. Little is, however, known about a role of central ghrelin in regulation of intestinal permeability. The present study was performed to clarify whether brain ghrelin is also involved in regulation of intestinal barrier function and its mechanism. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue in rats. Intracisternal injection of ghrelin dose-dependently abolished increased colonic permeability in response to LPS while intraperitoneal injection of ghrelin at the same dose or intracisternal injection of des-acyl-ghrelin failed to block it. Carbachol potently attenuated LPS-induced intestinal hyperpermeability, and atropine or bilateral subdiaphragmatic vagotomy prevented the improvement of intestinal hyperpermeability by central ghrelin. Intracisternal (D-Lys3)-GHRP-6, a selective ghrelin receptor antagonist, significantly blocked improvement of intestinal barrier function by intravenously administered 2-deoxy-d-glucose, central vagal stimulant. Intracisternal injection of orexin 1 receptor antagonist, SB-334867 blocked intracisternal ghrelin-induced improvement of colonic hyperpermeability. These results suggest that exogenously administered or endogenously released ghrelin acts centrally to improve a disturbed intestinal barrier function through orexinergic signaling and the vagal cholinergic pathway. Central ghrelin may be involved in the pathophysiology and be a novel therapeutic option in not only gastrointestinal diseases such as irritable bowel syndrome but also non-gastrointestinal diseases associated with the altered intestinal permeability.

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

Blocking VEGF by Bevacizumab Attenuates VEGF-Induced Vasospasm After Experimental Subarachnoid Hemorrhage in Rabbits

Vasospasm after subarachnoid hemorrhage (SAH) plays a vital role in the development of delayed cerebral ischemia. Anti- vascular endothelial growth factor (VEGF) antibodies, like bevacizumab (BEV), may attenuate VEGF-stimulated angiogenesis, reduced vascular cell proliferation, and improve vasospasm after SAH. Thirty-two adult male New Zealand white rabbits were randomly divided into 4 groups of 8 rabbits in each group: group 1 (control); group 2 (SAH); group 3 (SAH+ vehicle); and group 4 (SAH+ BEV). BEV (5 mg/kg, intraperitoneally) was administered 5 minutes after the intracisternal blood injection and continued for 72 hours once per day in the same dose for group 4. Animals were sacrificed 72 hours after SAH. Basilar artery cross-sectional areas, arterial wall thicknesses, and hippocampal degeneration scores were evaluated in all groups. VEGF is associated with the narrowing of the basilar artery. Treatment with BEV statistically significantly increased the cross-sectional area of the basilar artery when compared with the SAH and the vehicle groups. Basilar artery wall thicknesses in the BEV group was statistically significant smaller than in the SAH and vehicle groups. The hippocampal degeneration scores for the BEV and control groups were similar and significantly lower than those for the SAH and vehicle groups. Cellular proliferation and subsequent vessel wall thickening is a reason to delay cerebral ischemia and deterioration of the neurocognitive function. Intraperitoneal administration of BEV was found to attenuate cerebral vasospasm and prevent delayed cerebral ischemia and improve neurocognitive function after SAH in rabbits.

Adenosine A1 receptor agonist induces visceral antinociception via 5-HT1A, 5-HT2A, dopamine D1 or cannabinoid CB1 receptors, and the opioid system in the central nervous system

We have recently demonstrated that N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, acts centrally to induce a visceral antinociception. Since serotonin (5-HT), cannabinoid (CB), dopamine or opioid signaling in the central nervous system is involved in the regulation of visceral sensation, we made a hypothesis that the signaling may play a role in the CPA-induced visceral antinociception. Visceral sensation was evaluated by colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneously administered CPA significantly increased the threshold of colonic distension-induced AWR. Intracisternal injection of either 5-HT1A or 5-HT2A receptor antagonist blocked the CPA-induced visceral antinociception while 5-HT1B antagonist did not block the CPA-induced visceral antinociception. Subcutaneous injection of dopamine D1 receptor antagonist, CB1 receptor antagonist or naloxone significantly blocked the CPA-induced visceral antinociception while neither subcutaneous injection of dopamine D2 receptor antagonist nor CB2 receptor antagonist blocked the CPA-induced anti-pain action. These results suggest that 5-HT1A, 5-HT2A, dopamine D1, CB1 receptors and the opioid system in the CNS may specifically mediate the CPA-induced visceral antinociception. These findings may help in understanding the physiological relevance of central adenosine with special reference to the pathophysiology of altered visceral sensation especially in irritable bowel syndrome.