Reelin differentially shapes dendrite morphology of medial entorhinal cortical ocean and island cells.

The function of medial entorhinal cortex layer II (MECII) excitatory neurons has been recently explored. MECII dysfunction underlies deficits in spatial navigation and working memory. MECII neurons comprise two major excitatory neuronal populations, pyramidal island and stellate ocean cells, in addition to the inhibitory interneurons. Ocean cells express reelin and surround clusters of island cells that lack reelin expression. The influence of reelin expression by ocean cells and interneurons on their own morphological differentiation and that of MECII island cells has remained unknown. To address this, we used a conditional reelin knockout (RelncKO) mouse to induce reelin deficiency postnatally in vitro and in vivo. Reelin deficiency caused dendritic hypertrophy of ocean cells, interneurons and only proximal dendritic compartments of island cells. Ca2+ recording showed that both cell types exhibited an elevation of calcium frequencies in RelncKO, indicating that the hypertrophic effect is related to excessive Ca2+ signalling. Moreover, pharmacological receptor blockade in RelncKO mouse revealed malfunctioning of GABAB, NMDA and AMPA receptors. Collectively, this study emphasizes the significance of reelin in neuronal growth, and its absence results in dendrite hypertrophy of MECII neurons.

Green Synthesis and Characterization of Silver Nanoparticles Using Moringa Peregrina and Their Toxicity on MCF-7 and Caco-2 Human Cancer Cells.

Introduction: The synthesis of nanoparticles using naturally occurring reagents such as vitamins, sugars, plant extracts, biodegradable polymers and microorganisms as reductants and capping agents could be considered attractive for nanotechnology. These syntheses have led to the fabrication of limited number of inorganic nanoparticles. Among the reagents mentioned above, plant-based materials seem to be the best candidates, and they are suitable for large-scale biosynthesis of nanoparticles. Methods: The aqueous extract of Moringa peregrina leaves was used to synthesize silver nanoparticles. The synthesized nanoparticles were characterized by various spectral studies including FT-IR, SEM, HR-TEM and XRD. In addition, the antioxidant activity of the silver nanoparticles was studied viz. DPPH, ABTS, hydroxyl radical scavenging, superoxide radical scavenging, nitric oxide scavenging potential and reducing power with varied concentrations. The anticancer potential of the nanoparticles was also studied against MCF-7 and Caco-2 cancer cell lines. Results: The results showed that silver nanoparticles displayed strong antioxidant activity compared with gallic acid. Furthermore, the anticancer potential of the nanoparticles against MCF-7 and Caco-2 in comparison with the standard Doxorubicin revealed that the silver nanoparticles produced significant toxic effects against the studied cancer cell lines with the IC50 values of 41.59 (Caco-2) and 26.93 (MCF-7) µg/mL. Conclusion: In conclusion, the biosynthesized nanoparticles using M. peregrina leaf aqueous extract as a reducing agent showed good antioxidant and anticancer potential on human cancer cells and can be used in biological applications.

Unveiling the mechanisms of neuropathic pain suppression: perineural resiniferatoxin targets Trpv1 and beyond.

Neuropathic pain arises from damage or disorders affecting the somatosensory system. In rats, L5 nerve injury induces thermal and mechanical hypersensitivity/hyperalgesia. Recently, we demonstrated that applying resiniferatoxin (RTX) directly on uninjured L3 and L4 nerves alleviated thermal and mechanical hypersensitivity resulting from L5 nerve injury. Herein, using immunohistochemistry, Western blot, and qRT-PCR techniques, we reveal that perineural application of RTX (0.002%) on the L4 nerve substantially downregulated the expression of its receptor (Trpv1) and three different voltage-gated ion channels (Nav1.9, Kv4.3, and Cav2.2). These channels are found primarily in small-sized neurons and show significant colocalization with Trpv1 in the dorsal root ganglion (DRG). However, RTX treatment did not affect the expression of Kv1.1, Piezo2 (found in large-sized neurons without colocalization with Trpv1), and Kir4.1 (localized in satellite cells) in the ipsilateral DRGs. Furthermore, RTX application on L3 and L4 nerves reduced the activation of c-fos in the spinal neurons induced by heat stimulation. Subsequently, we investigated whether applying RTX to the L3 and L4 nerves 3 weeks before the L5 nerve injury could prevent the onset of neuropathic pain. Both 0.002 and 0.004% concentrations of RTX produced significant analgesic effects, while complete prevention of thermal and mechanical hypersensitivity required a concentration of 0.008%. Importantly, this preventive effect on neuropathic manifestations was not associated with nerve degeneration, as microscopic examination revealed no morphological changes. Overall, this study underscores the mechanisms and the significance of perineural RTX treatment applied to adjacent uninjured nerves in entirely preventing nerve injury-induced neuropathic pain in humans and animals.

Cutaneous Injection of Resiniferatoxin Completely Alleviates and Prevents Nerve-Injury-Induced Neuropathic Pain.

Fifth lumbar (L5) nerve injury in rodent produces neuropathic manifestations in the corresponding hind paw. The aim of this study was to investigate the effect of cutaneous injection of resiniferatoxin (RTX), a TRPV1 receptor agonist, in the rat's hind paw on the neuropathic pain induced by L5 nerve injury. The results showed that intraplantar injection of RTX (0.002%, 100 µL) (1) completely reversed the development of chronic thermal and mechanical hypersensitivity; (2) completely prevented the development of nerve-injury-induced thermal and mechanical hypersensitivity when applied one week earlier; (3) caused downregulation of nociceptive pain markers, including TRPV1, IB4 and CGRP, and upregulation of VIP in the ipsilateral dorsal horn of spinal cord and dorsal root ganglion (DRG) immunohistochemically and a significant reduction in the expression of TRPV1 mRNA and protein in the ipsilateral DRG using Western blot and qRT-PCR techniques; (4) caused downregulation of PGP 9.5- and CGRP-immunoreactivity in the injected skin; (5) produced significant suppression of c-fos expression, as a neuronal activity marker, in the spinal neurons in response to a second intraplantar RTX injection two weeks later. This work identifies the ability of cutaneous injection of RTX to completely alleviate and prevent the development of different types of neuropathic pain in animals and humans.

Primary Peripheral Epstein-Barr Virus Infection Can Lead to CNS Infection and Neuroinflammation in a Rabbit Model: Implications for Multiple Sclerosis Pathogenesis.

Epstein-Barr virus (EBV) is a common herpesvirus associated with malignant and non-malignant conditions. An accumulating body of evidence supports a role for EBV in the pathogenesis of multiple sclerosis (MS), a demyelinating disease of the CNS. However, little is known about the details of the link between EBV and MS. One obstacle which has hindered research in this area has been the lack of a suitable animal model recapitulating natural infection in humans. We have recently shown that healthy rabbits are susceptible to EBV infection, and viral persistence in these animals mimics latent infection in humans. We used the rabbit model to investigate if peripheral EBV infection can lead to infection of the CNS and its potential consequences. We injected EBV intravenously in one group of animals, and phosphate-buffered saline (PBS) in another, with and without immunosuppression. Histopathological changes and viral dynamics were examined in peripheral blood, spleen, brain, and spinal cord, using a range of molecular and histopathology techniques. Our investigations uncovered important findings that could not be previously addressed. We showed that primary peripheral EBV infection can lead to the virus traversing the CNS. Cell associated, but not free virus in the plasma, correlated with CNS infection. The infected cells within the brain were found to be B-lymphocytes. Most notably, animals injected with EBV, but not PBS, developed inflammatory cellular aggregates in the CNS. The incidence of these aggregates increased in the immunosuppressed animals. The cellular aggregates contained compact clusters of macrophages surrounded by reactive astrocytes and dispersed B and T lymphocytes, but not myelinated nerve fibers. Moreover, studying EBV infection over a span of 28 days, revealed that the peak point for viral load in the periphery and CNS coincides with increased occurrence of cellular aggregates in the brain. Finally, peripheral EBV infection triggered temporal changes in the expression of latent viral transcripts and cytokines in the brain. The present study provides the first direct in vivo evidence for the role of peripheral EBV infection in CNS pathology, and highlights a unique model to dissect viral mechanisms contributing to the development of MS.

Co-localization of nociceptive markers in the lumbar dorsal root ganglion and spinal cord of dromedary camel.

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa.

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.

Neuroprotective Effect of Curcumin on the Nigrostriatal Pathway in a 6-Hydroxydopmine-Induced Rat Model of Parkinson's Disease is Mediated by α7-Nicotinic Receptors.

Parkinson's disease (PD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic nigrostriatal neurons. Most of the existing pharmacological approaches in PD consider replenishing striatal dopamine. It has been reported that activation of the cholinergic system has neuroprotective effects on dopaminergic neurons, and human α7-nicotinic acetylcholine receptor (α7-nAChR) stimulation may offer a potential therapeutic approach in PD. Our recent in-vitro studies demonstrated that curcumin causes significant potentiation of the function of α7-nAChRs expressed in Xenopus oocytes. In this study, we conducted in vivo experiments to assess the role of the α7-nAChR on the protective effects of curcumin in an animal model of PD. Intra-striatal injection of 6-hydroxydopmine (6-OHDA) was used to induce Parkinsonism in rats. Our results demonstrated that intragastric curcumin treatment (200 mg/kg) significantly improved the abnormal motor behavior and offered neuroprotection against the reduction of dopaminergic neurons, as determined by tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra and caudoputamen. The intraperitoneal administration of the α7-nAChR-selective antagonist methyllycaconitine (1 µg/kg) reversed the neuroprotective effects of curcumin in terms of both animal behavior and TH immunoreactivity. In conclusion, this study demonstrates that curcumin has a neuroprotective effect in a 6-hydroxydopmine (6-OHDA) rat model of PD via an α7-nAChR-mediated mechanism.

Perineural application of resiniferatoxin on uninjured L3 and L4 nerves completely alleviates thermal and mechanical hypersensitivity following L5 nerve injury in rats.

Fifth lumbar (L5) nerve injury in rats causes neuropathic pain manifested with thermal and mechanical hypersensitivity in the ipsilateral hind paw. This study aimed to determine whether the elimination of unmyelinated primary afferents of the adjacent uninjured nerves (L3 and L4) would alleviate peripheral neuropathic pain. Different concentrations of capsaicin or its analog, resiniferatoxin (RTX), were applied perineurally on either the left L4 or L3 and L4 nerves in Wistar rats whose left L5 nerves were ligated and cut. The application of both capsaicin and RTX on the L4 nerve significantly reduced both thermal and mechanical hypersensitivity. However, only the application of RTX on both L3 and L4 nerves completely alleviated all neuropathic manifestations. Interestingly, responses to thermal and mechanical stimuli were preserved, despite RTX application on uninjured L3, L4, and L5 nerves, which supply the plantar skin in rats. Perineural application of RTX caused downregulation of TRPV1, CGRP, and IB4 binding and upregulation of VIP in the corresponding dorsal root ganglia (DRG) and the dorsal horn of the spinal cord. In comparison, VGLUT1 and NPY immunoreactivities were not altered. RTX application did not cause degenerative or ultrastructural changes in the treated nerves and corresponding DRGs. The results demonstrate that RTX induces neuroplasticity, rather than structural changes in primary afferents, that are responsible for alleviating hypersensitivity and chronic pain. Furthermore, this study suggests that treating uninjured adjacent spinal nerves may be used to manage chronic neuropathic pain following peripheral nerve injury.

Descriptive Analysis of Cerebral Arterial Vascular Architecture in Dromedary Camel (Camelus dromedarius).

The artiodactyl brain has multiple levels of vascular pooling and the rostral epidural rete mirabile (RERM) at its base. The current study is the first of its kind to precisely demonstrate the arterial vasculature of the dromedary brain, utilizing a new casting method with colored latex and epoxy paint. In total, 35 freshly slaughtered dromedary heads were injected with colored latex or colored epoxy paint prior to dissection in order to reveal cerebral vasculature; Ten processed heads were chemically digested with 5% potassium hydroxide to obtain hard casts of cerebral arteries and anastomosing structures. The outcomes of this study ascertain the distinct vascular features of dromedaries that set them apart from other artiodactyls. In addition to the RERM, the dromedary possesses a well-developed ophthalmic and chiasmatic rete. The dromedary is similar to giraffe, goat, cat and pig in the contribution of middle meningeal artery to the rete mirabile; however, dromedaries have several arteries emerging directly from the cerebral arterial circle that supply the choroid plexus and pineal gland. Additionally, dromedaries exhibit a dominant basilar system that dominates the blood supply to the medulla oblongata, pons, and cerebellum. In our study, we were able to graphically prove the lack of connection between the areas supplied by vertebrobasilar system and carotid system in the dromedary. Furthermore, the vertebral artery does not branch into the basilar artery; instead, it acts as a contributing vessel to the ventral spinal artery that later fuse to form the basilar artery. This study employed the new casting method to illustrate a new arterial source to RERM and the various anastomoses among arterial sources supplying the brain in the dromedary. These anastomoses play an important role in maintaining an uninterrupted cerebral blood supply, decreasing the vulnerability of the fragile brain against ischemia and stroke, as well as, play an important role in maintaining blood pressure and flow in long-necked dromedaries when they raise or lower their heads.

A single GABA neuron receives contacts from myelinated primary afferents of two adjacent peripheral nerves. A possible role in neuropathic pain.

GAD67-EGFP mice were used in a series of experiments to provide anatomical evidence for the role of the reduction in myelinated primary afferent input to GABA spinal neurons in the production of neuropathic pain following peripheral L5 nerve injury. First, we confirmed that L5 injury in these mice produced mechanical and thermal hyperalgesia in the ipsilateral foot. Second, we injected a mixture of cholera toxin subunit-B (CTb) and isolectin B4 (IB4) in the sciatic nerve to selectively label its myelinated and unmyelinated primary afferents. Results showed that primary afferents of sciatic nerve extend from L2-L6 spinal segments. Third, we determined the central terminations of myelinated primary afferents of L4 and L5 spinal nerves following CTb injection in either nerve. The myelinated primary afferents of both nerves terminated in the corresponding and two to three rostral spinal segments with some fibers descending to terminate in the segment caudal to the level at which they entered indicating an intermingling of their terminals at the dorsal horn of the spinal cord. Fourthly, we injected CTb in L5 nerve and CTb HRP-conjugate in L4 nerve. Confocal microscopy and subsequent image analyses showed that individual EGFP-labeled neurons in L4 segment receive myelinated primary afferent contacts from both L4 and L5 nerves. Eliminating inputs from L5 nerve following its injury would result in less involvement of spinal GABA neurons which would very likely initiate neuronal sensitization in L4 segment. This could lead to the development of hyperalgesia in response to the stimulation of the adjacent uninjured L4 nerve.

Neurocognitive Predictors of Clinical Improvement in Selective Serotonin Reuptake Inhibitor-Treated Adolescents with Major Depressive Disorder.

OBJECTIVES: Numerous studies have suggested cognitive deficits as consistently associated with adolescent depression. No study to date, however, has assessed neurocognitive predictors of selective serotonin reuptake inhibitor (SSRI) treatment response in adolescents with depression. This study examined neurocognitive tasks at baseline as predictors of clinical improvement with SSRI treatment (fluoxetine) at week 6 and 12 in an adolescent population. METHODS: Adolescents with depression were recruited from a child and adolescent psychiatry outpatient clinic at a university medical center. Twenty-four adolescents (mean age 14.8 years) with Major Depressive Disorder completed tasks of the Cambridge Neuropsychological Test Automated Battery, including visual memory, executive functioning, sustained attention, and impulsivity. Depression severity, measured by the Children's Depression Rating Scale-Revised (CDRS-R), was assessed at week 6 and 12 and clinical improvement was defined as percentage (%) change in CDRS-R from baseline. RESULTS: Clinical improvement is noted at both week 6 (mean % change in CDRS-R [M] = 46.8, standard deviation [SD] = 51.9) and week 12 (M = 87.9, SD = 57.2). Results reveal that less difficulty in sustained attention (p = 0.02), lower impulsivity (p = 0.00), and better planning (p = 0.04) at baseline were predictors of greater clinical improvement at week 6. Lower impulsivity at baseline remained significantly predictive of clinical improvement at week 12 (p = 0.01). CONCLUSION: Neurocognitive assessments could potentially help identify a subset of depressed adolescents who may not respond to conventional SSRI treatment and who may be better candidates for alternative or augmentation treatments.

Activation of the subthalamic nucleus suppressed by high frequency stimulation: A c-Fos immunohistochemical study.

Deep brain stimulation applied at high frequency (HFS) to the subthalamic nucleus (STN) is used to ameliorate the symptoms of Parkinson's disease. The mechanism by which this is achieved remains controversial. In particular, it is uncertain whether HFS has a suppressive or excitatory action locally within the STN. Brief exposure of rats to ether anesthesia evokes pathological burst firing and associated expression of the immediate early gene c-Fos in STN neurons. We used this ether model of STN activation to test the effect of a range of HFS parameters on c-Fos expression evoked by the anesthetic. The elevated baseline of c-Fos expression afforded the possibility of detecting further excitatory, or suppressive effects of STN HFS. Four HFS protocols were examined; 130, 200 and 260 Hz with 60 µs, and 130 Hz with 90 µs pulse width (HFS intensity:150-300 µA). All HFS protocols were applied for 20 min while the animals were exposed to ether. Ether-evoked expression of c-Fos immunoreactivity was suppressed by HFS at 200 and 260 Hz with a pulse width of 60 µs, and by 130 Hz when the pulse width was increased to 90 µs. HFS at 130 Hz with the 60 µs pulse width had no significant effect and HFS alone caused negligible c-Fos expression in the STN. These findings suggest that HFS of the STN causes significant suppression of evoked neuronal activity. It remains to be determined whether this locally suppressive property of HFS is associated with the efficacy of STN deep brain stimulation to relieve the symptoms of Parkinson's disease.

Increased pro-inflammatory cytokines, glial activation and oxidative stress in the hippocampus after short-term bilateral adrenalectomy.

BACKGROUND: Bilateral adrenalectomy has been shown to damage the hippocampal neurons. Although the effects of long-term adrenalectomy have been studied extensively there are few publications on the effects of short-term adrenalectomy. In the present study we aimed to investigate the effects of short-term bilateral adrenalectomy on the levels of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α; the response of microglia and astrocytes to neuronal cell death as well as oxidative stress markers GSH, SOD and MDA over the course of time (4 h, 24 h, 3 days, 1 week and 2 weeks) in the hippocampus of Wistar rats. RESULTS: Our results showed a transient significant elevation of pro-inflammatory cytokines IL-1ß and IL-6 from 4 h to 3 days in the adrenalectomized compared to sham operated rats. After 1 week, the elevation of both cytokines returns to the sham levels. Surprisingly, TNF-α levels were significantly elevated at 4 h only in adrenalectomized compared to sham operated rats. The occurrence of neuronal cell death in the hippocampus following adrenalectomy was confirmed by Fluoro-Jade B staining. Our results showed a time dependent increase in degenerated neurons in the dorsal blade of the dentate gyrus from 3 days to 2 weeks after adrenalectomy. Our results revealed an early activation of microglia on day three whereas activation of astroglia in the hippocampus was observed at 1 week postoperatively. A progression of microglia and astroglia activation all over the dentate gyrus and their appearance for the first time in CA3 of adrenalectomized rats hippocampi compared to sham operated was seen after 2 weeks of surgery. Quantitative analysis revealed a significant increase in the number of microglia (3, 7 and 14 days) and astrocytes (7 and 14 days) of ADX compared to sham operated rats. Our study revealed no major signs of oxidative stress until 2 weeks after adrenalectomy when a significant decrease of GSH levels and SOD activity as well as an increase in MDA levels were found in adrenalectomized compared to sham rats. CONCLUSION: Our study showed an early increase in the pro-inflammatory cytokines followed by neurodegeneration and activation of glial cells as well as oxidative stress. Taking these findings together it could be speculated that the early inflammatory components might contribute to the initiation of the biological cascade responsible for subsequent neuronal death in the current neurodegenerative animal model. These findings suggest that inflammatory mechanisms precede neurodegeneration and glial activation.

The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury.

Although repetitive Transcranial Magnetic Stimulation (rTMS) in treatment of stroke in humans has been explored over the past decade the data remain controversial in terms of optimal stimulation parameters and the mechanisms of rTMS long-term effects. This study aimed to explore the potential of different rTMS protocols to induce changes in gene expression in rat cortices after acute ischemic-reperfusion brain injury. The stroke was induced by middle cerebral artery occlusion (MCAO) with subsequent reperfusion. Changes in the expression of 96 genes were examined using low-density expression arrays after MCAO alone and after MCAO combined with 1Hz, 5Hz, continuous (cTBS) and intermittent (iTBS) theta-burst rTMS. rTMS over the lesioned hemisphere was given for two weeks (with a 2-day pause) in a single daily session and a total of 2400 pulses. MCAO alone induced significant upregulation in the expression of 44 genes and downregulation in 10. Two weeks of iTBS induced significant increase in the expression of 52 genes. There were no downregulated genes. 1Hz and 5Hz had no significant effects on gene expression, while cTBS effects were negligible. Upregulated genes included those involved in angiogenesis, inflammation, injury response and cellular repair, structural remodeling, neuroprotection, neurotransmission and neuronal plasticity. The results show that long-term rTMS in acute ischemic-reperfusion brain injury induces complex changes in gene expression that span multiple pathways, which generally promote the recovery. They also demonstrate that induced changes primarily depend on the rTMS frequency (1Hz and 5Hz vs. iTBS) and pattern (cTBS vs. iTBS). The results further underlines the premise that one of the benefits of rTMS application in stroke may be to prime the brain, enhancing its potential to cope with the injury and to rewire. This could further augment its potential to favorably respond to rehabilitation, and to restore some of the loss functions.

Anatomical evidence that the uninjured adjacent L4 nerve plays a significant role in the development of peripheral neuropathic pain after L5 spinal nerve ligation in rats.

Rats develop hyperalgesia and allodynia in the hind paw after L5 spinal nerve ligation. Phosphorylated extracellular regulated kinase (pERK) was used as a pain marker to investigate the potential role of adjacent uninjured L4 nerve in the development of heat hyperalgesia after L5 nerve injury. Left L5 nerve was ligated and sectioned in rats. Three days later, rats were randomly assigned to five groups; each had both hind paws immersed in water at different temperatures (no heat, 37, 42, 47, and 52 °C) under sevoflurane anesthesia for 2 minutes. Five minutes after stimulation the rats were sacrificed and sections of L3-L6 spinal segments were stained immunocytochemically with pERK antibody. pERK immunoreactivity, which is not detectable in the normal spinal cord, was discernible in neurons (not glia) of the superficial dorsal horn after noxious heat stimuli. pERK-positive neurons clearly overlapped in laminae I-II with normal unmyelinated and thin myelinated afferents labeled with calcitonin gene-related peptide and isolectin B4, and injured unmyelinated afferents labeled with vasoactive intestinal polypeptide. There was a linear increase in pERK immunoreactivity on both sides with an increase in temperature. Importantly, the number of positive pERK neurons was significantly higher in the ipsilateral side of L4 spinal segment, which receives innervation from uninjured L4 nerve, compared with the contralateral control side, which receives both uninjured L4 and L5 spinal nerves. The data demonstrate that the uninjured L4 nerve plays an important role in the development of heat hyperalgesia at the spinal cord level after L5 nerve injury.

Selective innervation of NK1 receptor-lacking lamina I spinoparabrachial neurons by presumed nonpeptidergic Aδ nociceptors in the rat.

Fine myelinated (Aδ) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to Aδ nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic Aδ nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r(+) and NK1r-lacking lamina I projection neurons are directly innervated by Aδ nociceptive afferents.

Fifth lumbar spinal nerve injury causes neurochemical changes in corresponding as well as adjacent spinal segments: a possible mechanism underlying neuropathic pain.

Previous investigations of the anatomical basis of the neuropathic-like manifestations in the spinal nerve ligation animal model have shown that the central terminations of the unmyelinated primary afferents of L5 spinal nerve are not restricted to the corresponding L5 spinal segment, and rather extend to two spinal segments rostrally and one segment caudally where they intermingle with primary afferents of the adjacent L4 spinal nerve. The aim of the present study was to investigate the neurochemical changes in the dorsal horn of the spinal cord and DRGs after L5 nerve injury in rats. In the first experiment, the right L5 nerve was ligated and sectioned for 14 days, and isolectin B4 (IB4, a tracer for unmyelinated primary afferents) was injected into the left L5 nerve. The results showed that the vasoactive intestinal peptide (VIP) was up-regulated in laminae I-II of L3-L6 spinal segments on the right side in exactly the same areas where IB4 labelled terminals were revealed on the left side. In the second experiment, L5 was ligated and sectioned and the spinal cord and DRGs were stained immunocytochemically with antibodies raised against various peptides known to be involved in pain transmission and hyperalgesia. The results showed that L5 nerve lesion caused down-regulation of substance P, calcitonin-gene related peptide and IB4 binding and up-regulation of neuropeptide Y and neurokinin-1 receptor in the dorsal horn of L4 and L5 spinal segments. Similar neurochemical changes were observed only in the corresponding L5 DRG with minimal effects observed in L3, L4 and L6 DRGs. Although, L5 nerve injury caused an up-regulation in NPY, no change in SP and CGRP immunoreactivity was observed in ipsilateral garcile nucleus. These neuroplastic changes in the dorsal horn of the spinal cord, in the adjacent uninjured territories of the central terminations of the adjacent uninjured nerves, might explain the mechanism of hyperalgesia after peripheral nerve injury.

Anticonvulsant properties of histamine H3 receptor ligands belonging to N-substituted carbamates of imidazopropanol.

Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. Therefore, the previously described and structurally strongly related imidazole-based derivatives belonging to carbamate class with high H3R in vitro affinity, in-vivo antagonist potency, and H3R selectivity profile were investigated on their anticonvulsant activity in maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-kindled seizure models in Wistar rats. The effects of systemic injection of H3R ligands 1-13 on MES-induced and PTZ-kindled seizures were screened and evaluated against the reference antiepileptic drug (AED) Phenytoin (PHT) and the standard histamine H3R inverse agonist/antagonist Thioperamide (THP) to determine their potential as new antiepileptic drugs. Following administration of the H3R ligands 1-13 (5, 10 and 15 mg/kg, ip) there was a significant dose dependent reduction in MES-induced seizure duration. The protective action observed for the pentenyl carbamate derivative 4, the most protective H3R ligand among 1-13, was significantly higher (P <0.05) than that of standard H3R antagonist THP, and was reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine (RAMH) (10mg/kg), or with the CNS penetrant H1R antagonist Pyrilamine (PYR) (10mg/kg). In addition, subeffective dose of H3R ligand 4 (5mg/kg, ip) significantly potentiated the protective action in rats pretreated with PHT (5mg/kg, ip), a dose without appreciable protective effect when given alone. In contrast, pretreatment with H3R ligand 4 (10mg/kg ip) failed to modify PTZ-kindled convulsion, whereas the reference drug PHT was found to fully protect PTZ-induced seizure. These results indicate that some of the investigated imidazole-based H3R ligands 1-13 may be of future therapeutic value in epilepsy.