Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.

A microchannel neuroprosthesis for bladder control after spinal cord injury in rat

A severe complication of spinal cord injury is loss of bladder function (neurogenic bladder), which is characterized by loss of bladder sensation and voluntary control of micturition (urination), and spontaneous hyperreflexive voiding against a closed sphincter (detrusor-sphincter dyssynergia). A sacral anterior root stimulator at low frequency can drive volitional bladder voiding, but surgical rhizotomy of the lumbosacral dorsal roots is needed to prevent spontaneous voiding and dyssynergia. However, rhizotomy is irreversible and eliminates sexual function, and the stimulator gives no information on bladder fullness. We designed a closed-loop neuroprosthetic interface that measures bladder fullness and prevents spontaneous voiding episodes without the need for dorsal rhizotomy in a rat model. To obtain bladder sensory information, we implanted teased dorsal roots (rootlets) within the rat vertebral column into microchannel electrodes, which provided signal amplification and noise suppression. As long as they were attached to the spinal cord, these rootlets survived for up to 3 months and contained axons and blood vessels. Electrophysiological recordings showed that half of the rootlets propagated action potentials, with firing frequency correlated to bladder fullness. When the bladder became full enough to initiate spontaneous voiding, high-frequency/amplitude sensory activity was detected. Voiding was abolished using a high-frequency depolarizing block to the ventral roots. A ventral root stimulator initiated bladder emptying at low frequency and prevented unwanted contraction at high frequency. These data suggest that sensory information from the dorsal root together with a ventral root stimulator could form the basis for a closed-loop bladder neuroprosthetic. Copyright © 2013, American Association for the Advancement of Science

Motor imagery-induced EEG patterns in individuals with spinal cord injury and their impact on brain–computer interface accuracy

Objective. Assimilating the diagnosis complete spinal cord injury (SCI) takes time and is not easy, as patients know that there is no ‘cure’ at the present time. Brain–computer interfaces (BCIs) can facilitate daily living. However, inter-subject variability demands measurements with potential user groups and an understanding of how they differ to healthy users BCIs are more commonly tested with. Thus, a three-class motor imagery (MI) screening (left hand, right hand, feet) was performed with a group of 10 able-bodied and 16 complete spinal-cord-injured people (paraplegics, tetraplegics) with the objective of determining what differences were present between the user groups and how they would impact upon the ability of these user groups to interact with a BCI. Approach. Electrophysiological differences between patient groups and healthy users are measured in terms of sensorimotor rhythm deflections from baseline during MI, electroencephalogram microstate scalp maps and strengths of inter-channel phase synchronization. Additionally, using a common spatial pattern algorithm and a linear discriminant analysis classifier, the classification accuracy was calculated and compared between groups. Main results. It is seen that both patient groups (tetraplegic and paraplegic) have some significant differences in event-related desynchronization strengths, exhibit significant increases in synchronization and reach significantly lower accuracies (mean (M) = 66.1%) than the group of healthy subjects (M = 85.1%). Significance. The results demonstrate significant differences in electrophysiological correlates of motor control between healthy individuals and those individuals who stand to benefit most from BCI technology (individuals with SCI). They highlight the difficulty in directly translating results from healthy subjects to participants with SCI and the challenges that, therefore, arise in providing BCIs to such individuals

Motor imagery induced EEG patterns in spinal cord injury patients and their impact on Brain-Computer Interface accuracy

OBJECTIVE: Assimilating the diagnosis complete spinal cord injury (SCI) takes time and is not easy, as patients know that there is no 'cure' at the present time. Brain-computer interfaces (BCIs) can facilitate daily living. However, inter-subject variability demands measurements with potential user groups and an understanding of how they differ to healthy users BCIs are more commonly tested with. Thus, a three-class motor imagery (MI) screening (left hand, right hand, feet) was performed with a group of 10 able-bodied and 16 complete spinal-cord-injured people (paraplegics, tetraplegics) with the objective of determining what differences were present between the user groups and how they would impact upon the ability of these user groups to interact with a BCI. APPROACH: Electrophysiological differences between patient groups and healthy users are measured in terms of sensorimotor rhythm deflections from baseline during MI, electroencephalogram microstate scalp maps and strengths of inter-channel phase synchronization. Additionally, using a common spatial pattern algorithm and a linear discriminant analysis classifier, the classification accuracy was calculated and compared between groups. MAIN RESULTS: It is seen that both patient groups (tetraplegic and paraplegic) have some significant differences in event-related desynchronization strengths, exhibit significant increases in synchronization and reach significantly lower accuracies (mean (M) = 66.1%) than the group of healthy subjects (M = 85.1%). SIGNIFICANCE: The results demonstrate significant differences in electrophysiological correlates of motor control between healthy individuals and those individuals who stand to benefit most from BCI technology (individuals with SCI). They highlight the difficulty in directly translating results from healthy subjects to participants with SCI and the challenges that, therefore, arise in providing BCIs to such individuals.

Neurodegeneration and Increased Production of Nitrotyrosine, Nitric Oxide Synthase, IFN-gamma and S100 beta Protein in the Spinal Cord of IL-12p40-Deficient Mice Infected with Trypanosoma cruzi

Background/Aim: Chagas` disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100 beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100 beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100 beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p<0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p<0.01) and NOS (544%, p<0.001). Moreover, the intensity of nitrotyrosine (16%, p<0.01), NOS (38%, p<0.05) and S100 beta (21%, p<0.001) immunoreactivities were also augmented. No IFN-gamma labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled.Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100 beta may contribute to NOS activation in the absence of IL-12. Copyright (C) 2009 S. Karger AG, Basel

Canine spinal cord neuron and axon myelin sheath morphometry

This inedited morphometric study has been developed from healthy canine spinal cord neuron cytoplasm and nucleus, and white matter axonal myelin sheath, from cervical, thoracic and lumbar regions. For the morphometric study, the parameters were area, perimeter, maximum and minimum diameters and roundness for neurons and myelin thickness for axon. For each parameter, 300 neurons were analysed. The results revealed that lumbar neurons had the highest mean values for the analysed parameters, indicating the presence of large neurons in this region, with large axons as a result of myelin thickness, which is proportional to axon calibre. We conclude that these morphometric results can contribute for the establishment of normal patterns, for canine spinal cord cervical, thoracic and lumbar segments.

The effects of intrathecal administration of betamethasone over the dogs' spinal cord and meninges

OBJETIVO: Determinar possíveis alterações clínicas e histológicas determinadas pela administração da betametasona no espaço subaracnóideo de cães. MÉTODOS: Vinte e um cães foram incluídos no estudo de forma aleatória e encoberta. Depois de anestesiados, os cães foram submetidos a punção subaracóidea com injeção de 1 ml da solução sorteada. Os animais receberam solução salina 0,9% em G1, betametasona na dose de 1,75 mg em G2 e betametasona na dose de 3,5 mg em G3. Todos os animais foram mantidos em observação clínica por 21 dias, sendo posteriormente sacrificados. Porções da medula espinhal e sacral foram removidas para análise histológica por microscopia óptica. RESULTADOS: Não foram detectadas alterações clínicas em quaisquer dos animais incluídos no estudo. da mesma forma, nenhum animal do G1 apresentou alterações histológicas. Infiltração inflamatória foi observada em dois cães, um do G2 e outro e G3. No cão do G2 onde a infiltração inflamatória foi observada ocorreu, conjuntamente, hemorragia e necrose. em dois cães, um de G2 e outro de G3, observou-se discreta fibrose e espessamento da aracnóide, sendo focal em um e difusa no outro. CONCLUSÃO: A administração subaracnóidea de betametasona determinou alterações histológicas em medula e meninges de alguns dos cães envolvidos no estudo.

The Effects of Subarachnoid Administration of Preservative-Free S(+)-Ketamine on Spinal Cord and Meninges in Dogs

BACKGROUND: The N-methyl-D-aspartate receptor antagonist ketamine and its active enantiomer, S(+)-ketamine, have been injected in the epidural and subarachnoid spaces to treat acute postoperative pain and relieve neuropathic pain syndrome. In this study we evaluated the effects of a single dose of preservative-free S(+)-ketamine, in doses usually used in clinical practice, in the spinal cord and meninges of dogs.METHODS: Under anesthesia (IV etomidate (2 mg/kg) and fentanyl (0.005 mg/kg), 16 dogs (6 to 15 kg) were randomized to receive a lumbar intrathecal injection (L5/6) of saline solution of 0.9% (control group) or S(+)-ketamine 1 mg/kg(-1) (ketamine group). All doses were administered in a volume of 1 mL over a 10-second interval. Accordingly, injection solution ranged from 0.6% to 1.5%. After 21 days of clinical observation, the animals were killed; spinal cord, cauda equine root, and meninges were removed for histological examination with light microscopy. Tissues were examined for demyelination (Masson trichrome), neuronal death (hematoxylin and eosin) and astrocyte activation (glial fibrillary acidic protein).RESULTS: No clinical or histological alterations of spinal tissue or meninges were found in animals from either control or ketamine groups.CONCLUSION: A single intrathecal injection of preservative-free S(+)-ketamine, at 1 mg/kg-1 dosage, over a concentration range of 6 to 15 mg/mL injected in the subarachnoid space in a single puncture, did not produce histological alterations in this experimental model. (Anesth Analg 2012;114:450-55)

Respiratory rhythm of brainstem-spinal cord preparations: Effects of maturation, age, mass and oxygenation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects caused by the spinal administration of ketamine S (+) 5% with no preservatives, using a single puncture, and located on the spinal cord and meninges in rabbits

PURPOSE: To evaluate the effect of ketamine S (+) 5% with no preservatives and administered as a subarachnoid single puncture on the spinal cord and meninges of rabbits.METHODS: Twenty young adult female rabbits, each weighing 3500-5000 g and having a spine length between 34 and 38 cm, were divided by lot into two groups (G): 0.9% saline in G1 and ketamine S (+) 5% in G2, by volume of 5 μg per cm column (0.18 mL). After intravenous anaesthesia with ketamine and xylazine, the subarachnoid space was punctured at S1-S2 under ultrasound guidance, and a random solution was injected. The animals remained in captivity for 21 days under medical observation and were sacrificed by decapitation. The lumbosacral spinal cord portion was removed for immunohistochemistry to assess the glial fibrillary acidic protein (GFAP), and histology was assessed using hematoxylin and eosin (HE) stain.RESULTS:No histological lesions were found in the nervous tissue (roots and cord) or meninges in either group.CONCLUSION: The ketamine S (+) 5% unpreserved triggered no neurological or histological lesions in the spinal cord or meninges of rabbits.

Low-intensity laser therapy effect on the recovery of traumatic spinal cord injury

Scientific advances have been made to optimize the healing process in spinal cord injury. Studies have been developed to obtain effective treatments in controlling the secondary injury that occurs after spinal cord injury, which substantially changes the prognosis. Low-intensity laser therapy (LILT) has been applied in neuroscience due to its anti-inflammatory effects on biological tissue in the repairing process. Few studies have been made associating LILT to the spinal cord injury. The objective of this study was to investigate the effect of the LILT (GaAlAs laser-780 nm) on the locomotor functional recovery, histomorphometric, and histopathological changes of the spinal cord after moderate traumatic injury in rats (spinal cord injury at T9 and T10). Thirty-one adult Wistar rats were used, which were divided into seven groups: control without surgery (n = 3), control surgery (n = 3), laser 6 h after surgery (n = 5), laser 48 h after surgery (n = 5), medullar lesion (n = 5) without phototherapy, medullar lesion + laser 6 h after surgery (n = 5), and medullar lesion + laser 48 h after surgery (n = 5). The assessment of the motor function was performed using Basso, Beattie, and Bresnahan (BBB) scale and adapted Sciatic Functional Index (aSFI). The assessment of urinary dysfunction was clinically performed. After 21 days postoperative, the animals were euthanized for histological and histomorphometric analysis of the spinal cord. The results showed faster motor evolution in rats with spinal contusion treated with LILT, maintenance of the effectiveness of the urinary system, and preservation of nerve tissue in the lesion area, with a notorious inflammation control and increased number of nerve cells and connections. In conclusion, positive effects on spinal cord recovery after moderate traumatic spinal cord injury were shown after LILT.

Contribution of magnetic resonance in acute spinal cord trauma - case report

Acute spinal cord trauma is a common injury that occurs frequently in small animals. In order to acertain a prognosis of the lesion generated in the spinal cord, it is necessary to perform a complete neurological and physical examination, aided by complementary images. Magnetic resonance imaging may be advantageous over other types of images, because it can determine with greater definition the structural damage to the nervous tissue. The objective of this report was to demonstrate the contribution of magnetic resonance imaging in a case of acute spinal cord trauma in a dog.

Toxoplasmosis: morphological and morphometric evaluation of spinal cord neurons from nonsymptomatic seropositive dogs

The aim of this work was to analyze the neuron morphology and morphometry of cervical, thoracic and lumbar areas of nonsymptomatic seropositive dogs’ spinal cord for toxoplasmosis. Twenty indefinite-breed adult dogs were used; ten dogs were healthy, with negative serology for toxoplasmosis, and were used as the control group (group 1), and ten dogs were nonsymptomatic but seropositive for toxoplasmosis (group 2). After the microtomy, with interval of 100 micrometers (µm), the histological 5-µm-thick cuts were dyed by hematoxylin-eosin and Masson's trichrome techniques. The glass slides were analyzed under light microscope to examine the neuron morphology. The parameters considered for the morphometric analysis were area, perimeter, maximum diameter, minimum diameter and shape factor of cytoplasm and nucleus of neuron. The results were statistically analyzed by Student’s t test at 5% probability level. The morphological characteristics between the two groups were similar and according to literature. The morphometric results showed that there were changes in neurons size and structure, and increase and loss of star shape were noticed in seropositive animals. The results suggest that the neurons of these dogs, yet nonsymptomatic, can have lost their conductor function.

Effect of mesenchymal stem cells on movement and urination of rats with spinal cord injury

Cell therapy has frequently been reported as a possible treatment for spinal trauma in humans and animals; however, without pharmacologically curative action on damage from the primary lesion. In this study, we evaluated the effect of administering human adipose-derived stem cells (hADSC) in rats after spinal cord injury. The hADSC were used between the third and fifth passages and a proportion of cells were transduced for screening in vivo after transplantation. Spinal cord injury was induced with a Fogarty catheter no. 3 inserted into the epidural space with a cuff located at T8 and filled with 80 mu L saline for 5 min. The control group A (n = 12) received culture medium (50 mu L) and group B (n = 12) received hADSC (1.2 x 10(6)) at 7 and 14 days post-injury, in the tail vein. Emptying of the bladder by massage was performed daily for 3 months. Evaluation of functional motor activity was performed daily until 3 months post-injury using the Basso-Beattie-Bresnahan scale. Subsequently, the animals were euthanized and histological analysis of the urinary bladder and spinal cord was performed. Bioluminescence analysis revealed hADSC at the application site and lungs. There was improvement of urinary bladder function in 83.3% animals in group B and 16.66% animals in group A. The analysis of functional motor activity and histology of the spinal cord and urinary bladder demonstrated no significant difference between groups A and B. The results indicate that transplanted hADSC improved urinary function via a telecrine mechanism, namely action at a distance.