Spinal Sections Research Articles

Anatomy of the spinal dorsal root entry zone: its clinical significance.

The posterolateral sulcus (PLS) is an important surgical landmark, especially for DREZ (dorsal root entry zone) operations. The present study aimed to show the variations of the PLS using human spinal cord histological sections and report the variability in the number of dorsal rootlets of the spinal nerves in each the spinal cord segment. Further, measure the height and width of the dorsal horn on histological sections for cervical, thoracic, and lumbar levels. The results of the present study showed various patterns of PLS 1.clearly present PLS, 2. short PLS, 3. absent PLS or 4. irregular PLS. Height and width measurements of the dorsal horn showed that the average width was greatest at lower cervical (0.48 ± 0.04 mm) and least at lower thoracic levels (0.41 ± 0.04 mm), whereas the average height was greatest at upper cervical (3.0 ± 0.06 mm) and smallest at lower lumbar levels (1.8 ± 0.08 mm). The average number of rootlets varied considerably, at cervical level it was 7.6 ± 1.4 mm, at thoracic 6.6 ± 0.8 mm and at lumbar 6.1 ± 0.4 mm. The detailed anatomy of the variations of the PLS and the average number of rootlets at each spinal level can increase the success of regional surgery. Further, fine measurements on histological sections can give detailed knowledge on the size necessary for lesioning in DREZ operations.

Impact depth and the interaction with impact speed affect the severity of contusion spinal cord injury in rats.

Spinal cord injury (SCI) biomechanics suggest that the mechanical factors of impact depth and speed affect the severity of contusion injury, but their interaction is not well understood. The primary aim of this work was to examine both the individual and combined effects of impact depth and speed in contusion SCI on the cervical spinal cord. Spinal cord contusions between C5 and C6 were produced in anesthetized rats at impact speeds of 8, 80, or 800 mm/s with displacements of 0.9 or 1.5 mm (n=8/group). After 7 days postinjury, rats were assessed for open-field behavior, euthanized, and spinal cords were harvested. Spinal cord tissue sections were stained for demyelination (myelin-based protein) and tissue sparing (Luxol fast blue). In parallel, a finite element model of rat spinal cord was used to examine the resulting maximum principal strain in the spinal cord during impact. Increasing impact depth from 0.9 to 1.5 mm reduced open-field scores (p<0.01) above 80 mm/s, reduced gray (GM) and white matter (WM) sparing (p<0.01), and increased the amount of demyelination (p<0.01). Increasing impact speed showed similar results at the 1.5-mm impact depth, but not the 0.9-mm impact depth. Linear correlation analysis with finite element analysis strain showed correlations (p<0.001) with nerve fiber damage in the ventral (R(2)=0.86) and lateral (R(2)=0.74) regions of the spinal cord and with WM (R(2)=0.90) and GM (R(2)=0.76) sparing. The results demonstrate that impact depth is more important in determining the severity of SCI and that threshold interactions exist between impact depth and speed.

Highly sensitive visualization of inorganic mercury in mouse neurons using a fluorescent probe.

In the present study, we used the previously developed fluorescence probe, EPNP, to generate the first image of the distribution of mercuric ion in primary mouse neuron cultures. At postnatal day 1 (P1), the mice were intraperitoneally (IP) injected with mercuric chloride in doses ranging from 0.05 to 0.6 μg/g body weight. After 1, 2, 3, and 4 days exposure, primary nervous cell cultures and frozen brain and spinal tissue sections were prepared and dyed using EPNP. On the third day of repeated injections, Hg(2+) was visualized in primary cerebral neuron cultures as an increase of Hg(2+)-induced fluorescence at the doses ≥ 0.1 μg/g. A similar accumulation of Hg(2+) was observed in frozen hippocampus tissue sections. In contrast, no Hg(2+) was observed in spinal cord neurons and spinal tissue sections. The detection of a low dose of IP injected mercury in mouse cerebral neurons facilitated the evaluation of the exposure risk to low-dose Hg(2+) in immature organisms. Moreover, the highly sensitive EPNP revealed Hg(2+) in the cerebral neurons of mice younger than P4, while the presence of Hg(2+) was not detected until ≥ P11 in previous reports. Thus, this technology and the results obtained herein are of interest for neurotoxicology.

Экспериментальная полная транссекция спинного мозга и его биоинженерная реконструкция

The work represents the original research devoted to cellular and issue reconstruction of a spinal cord injury in adult rats with an experimental vertebral-spinal trauma. After performing a laminectomy, partial and full mechanical intersection of the spinal cord at the IX thoracic vertebrae in the spinal implanted medical devices containing bovine collagen and crab chitosan, sulfated and non-sulfated glycosaminoglycans, full nutrient medium, conditioned nutrient medium with neurotrophic factors in the growth of brain cells mouse embryos and embryonic mouse stem cells, N2 neuronal additive, retinoic acid. The dynamics neurologic status in animals has shown essential reduction of deficiency on BBB scale during 20 weeks of the postoperative period. The indirect immune-fluorescence method of a spinal cord cells and histological sections confirms presence of active introduction of cells of a parent spinal cord into implant, high viability of the replaced cells of the mouse during all period of supervision, formation in 1 week after operation of progenitor neuronal cells with expression of neurotransmitters. This change is accompanied by a partial recovery of motor, sensory and vegetative functions of the spinal cord, reduction in the level of neuro-deficit of 5, 6 points on a scale of BBB. Transplantation of collagen-chitosan matrix containing 100 thousand cells, neuronal precursors leads for 20 weeks of observation to preserve their viability, formation of numerous neurons, creating intra-synaptic correlation associated to early expression of neurotransmitters, significant restoration of motor and sensory functions of the spinal cord, reaching a level of reduction neuro-deficit equal to 19.5 points on a scale of BBB.

PD-L1 is increased in the spinal cord and infiltrating lymphocytes in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis is a mouse model of human multiple sclerosis with similar pathology and pathogenesis. Th1 cells play an important role in the pathogenesis of experimental allergic encephalomyelitis. This study determined the potential effect of programmed cell death 1 ligand 1 in the pathogenesis of experimental allergic encephalomyelitis induced by injecting myelin oligodendrocyte glycoprotein, complete Freund's adjuvant and Bordetella pertussis toxin into C57BL/6J mice. Experimental allergic encephalomyelitis mice developed disease and showed inflammatory changes in the central nervous system by hematoxylin-eosin staining of spinal cord pathological sections, demyelination by Luxol fast-blue staining and clinical manifestations. The expression of programmed cell death 1 ligand 1 in mice was detected by immunohistochemistry, flow cytometry and western blot analysis. The expression of programmed cell death 1 ligand 1 in the spinal cord and splenocytes of mice was significantly increased compared with normal mice. Our findings suggest the involvement of programmed cell death 1 ligand 1 in the pathogenesis of experimental allergic encephalomyelitis and suggest this should be studied in multiple sclerosis.

Tacolimus postconditioning alleviates apoptotic cell death in rats after spinal cord ischemia-reperfusion injury via up-regulating protein-serine-threonine kinases phosphorylation

The effects of tacrolimus postconditioning on protein-serine-threonine kinases (Akt) phosphorylation and apoptotic cell death in rats after spinal cord ischemia-reperfusion injury were investigated. Ninety male SD rats were randomly divided into sham operation group, ischemia-reperfusion group and tacrolimus postconditioning group. The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation. The spinal cord was reperfused 20 min after ischemia via removing saline out of balloon. The corresponding spinal cord segments were excised and determined for Akt activity in spinal cord tissue by using Western blotting at 5, 15, and 60 min after reperfusion respectively. Spinal cord tissue sections were stained immunohistochemically for detection of the phosphorylated Akt expression at 15 min after reperfusion. Flow cytometry was applied to assess apoptosis of neural cells, and dry-wet weights method was employed to measure water content in spinal cord tissue at 24 h after reperfusion. The results showed that the activities of Akt in tarcolimus postconditioning group were significantly higher than those in ischemia-reperfusion group at 5, 15, and 60 min after reperfusion (P<0.05, P<0.01). The Akt activities reached the peak at 15 min after reperfusion in ischemia-reperfusion group and tacrolimus postconditioning group. The percentage of apoptotic cells and water content in spinal cord tissue were significantly reduced (P<0.01) in tacrolimus postconditioning group as compared with those in ischemia-reperfusion group at 24 h after reperfusion. It is concluded that tacrolimus post-conditioning can increase Akt activity in spinal cord tissue of rats, inhibit apoptosis of neural cells as well as tissue edema, and thereby alleviate spinal cord ischemia-reperfusion injury.

Effects of treating traumatic brain injury with collagen scaffolds and human bone marrow stromal cells on sprouting of corticospinal tract axons into the denervated side of the spinal cord

This study was designed to investigate how transplantation into injured brain of human bone marrow stromal cells (hMSCs) impregnated in collagen scaffolds affects axonal sprouting in the spinal cord after traumatic brain injury (TBI) in rats. Also investigated was the relationship of axonal sprouting to sensorimotor functional recovery after treatment. Adult male Wistar rats (n = 24) underwent a controlled cortical impact injury and were divided into three equal groups (8 rats/group). The two treatment groups received either hMSCs (3 × 10(6)) alone or hMSC (3 × 10(6))-impregnated collagen scaffolds transplanted into the lesion cavity. In the control group, saline was injected into the lesion cavity. All treatments were performed 7 days after TBI. On Day 21 after TBI, a 10% solution of biotinylated dextran amine (10,000 MW) was stereotactically injected into the contralateral motor cortex to label the corticospinal tract (CST) originating from this area. Sensorimotor function was tested using the modified neurological severity score (mNSS) and foot-fault tests performed on Days 1, 7, 14, 21, 28, and 35 after TBI. Spatial learning was tested with Morris water maze test on Days 31-35 after TBI. All rats were sacrificed on Day 35 after TBI, and brain and spinal cord (cervical and lumbar) sections were stained immunohistochemically for histological analysis. Few biotinylated dextran amine-labeled CST fibers crossing over the midline were found in the contralateral spinal cord transverse sections at both cervical and lumbar levels in saline-treated (control) rats. However, hMSC-alone treatment significantly increased axonal sprouting from the intact CST into the denervated side of the gray matter of both cervical and lumbar levels of the spinal cord (p < 0.05). Also, this axonal sprouting was significantly more in the scaffold+hMSC group compared with the hMSC-alone group (p < 0.05). Sensorimotor functional analysis showed significant improvement of mNSS (p < 0.05) and foot-fault tests (p < 0.05) in hMSC-alone and scaffold+hMSC-treated rats compared with controls (p < 0.05). Functional improvement, however, was significantly greater in the scaffold+hMSC group compared with the hMSC-alone group (p < 0.05). Morris water maze testing also showed significant improvement in spatial learning in scaffold+hMSC and hMSC-alone groups compared with the control group (p < 0.05), with rats in the scaffold+hMSC group performing significantly better than those in the hMSC-alone group (p < 0.05). Pearson correlation data showed significant correlation between the number of crossing CST fibers detected and sensorimotor recovery (p < 0.05). Axonal plasticity plays an important role in neurorestoration after TBI. Transplanting hMSCs with scaffolds enhances the effect of hMSCs on axonal sprouting of CST fibers from the contralateral intact cortex into the denervated side of spinal cord after TBI. This enhanced axonal regeneration may at least partially contribute to the therapeutic benefits of treating TBI with hMSCs.

Automatic identification and quantitative morphometry of unstained spinal nerve using molecular hyperspectral imaging technology

Quantitative observation of nerve fiber sections is often complemented by morphological analysis in both research and clinical condition. However, existing manual or semi-automated methods are tedious and labour intensive, fully automated morphometry methods are complicated as the information of color or gray images captured by traditional microscopy is limited. Moreover, most of the methods are time-consuming as the nerve sections need to be stained with some reagents before observation. To overcome these shortcomings, a molecular hyperspectral imaging system is developed and used to observe the spinal nerve sections. The molecular hyperspectral images contain both the structural and biochemical information of spinal nerve sections which is very useful for automatic identification and quantitative morphological analysis of nerve fibers. This characteristic makes it possible for researchers to observe the unstained spinal nerve and live cells in their native environment. To evaluate the performance of the new method, the molecular hyperspectral images were captured and the improved spectral angle mapper algorithm was proposed and used to segment the myelin contours. Then the morphological parameters such as myelin thickness and myelin area were calculated and evaluated. With these morphological parameters, the three dimension surface view images were drawn to help the investigators observe spinal nerve at different angles. The experiment results show that the hyperspectral based method has the potential to identify the spinal nerve more accurate than the traditional method as the new method contains both the spectral and spatial information of nerve sections.

Mo1848 Characterization of Dorsal Horn Neurons Activated by Noxious Colorectal Distension in Health and Chronic Visceral Hypersensitivity

Background: Little is known about the spinal cord dorsal horn (DH) neurons activated by colonic mechanical stimuli. Even less is known about how chronic hypersensitivity of colonic nociceptors effects DH neuron activity. Methods: Experiments were performed in healthy mice (N=4) and in a TNBS model of post-inflammatory chronic visceral hypersensitivity (CVH; N=4). TNBS (130μL/ml) was administered by colorectal enema and after 28 days mice underwent 80 mmHg of colorectal distension (CRD). By this time inflammation is resolved and the peripheral endings of colonic nociceptors are mechanically hypersensitive1. Following CRD, mice were perfused fixed, spinal cord T10-L1 removed and processed for phosphorylated MAP kinase ERK 1/2 (pERK) immunohistochemistry to identify DH neurons activated by CRD. Spinal sections were co-labeled for calcitonin gene related peptide (CGRP), isolectin B4 (IB4), calbindin, GABA and NMDA receptor subunit 2B (NMDAr2B). The average number of pERK-immunoreactive (IR) neurons from 6-10 spinal sections was compared between healthy and post-inflamed mice using Two-way ANOVA with Bonferroni's posttests whilst unpaired student t-tests determined differences in co-labelling. Results: Significantly more DH neurons were pERK-IR following CRD in CVH mice compared to healthy mice (P<0.0001), specifically in T12-T13 (P<0.05) and T13-L1 (P<0.001). In healthy mice, pERK-IR neurons were located in CGRP-IR laminae I (LI), but not in the IB4-IR substantia gelatinosa. 37±3% of pERK-IR neurons were calbindin-IR, which accounted for 3±0.8% of all calbindin-IR DH neurons, classified as projection neurons in LI and a subtype of excitatory interneuron in LI-LII. All pERK/calbindin-IR neurons were located in LI and NMDAr2B-IR. 3.8±2% of pERK-IR neurons were GABA-IR, which accounted for 0.9±0.6% of all GABAIR DH inhibitory interneurons. In CVH mice, pERK-IR neurons were located in CGRP-IR LI and ventral to the IB4-IR substantia gelatinosa. A smaller proportion of pERK-IR neurons were calbindin-IR (14±5%, P=0.07), which was a consequence of more pERK-IR neurons not containing calbindin-immunoreactivity and the number of pERK/calbindin-IR neurons unchanged. Significantly more pERK-IR neurons were GABA-IR in CVH mice (13.45±3%, P<0.05), with pERK-IR neurons accounting for a greater number of GABA-IR neurons (8±1%, P<0.05). Conclusion: We identified DH neurons responsive to colonic mechanical stimulation. We also show that the chronic hypersensitivity of colonic afferent endings following inflammation leads to a significant increase in the number of DHneurons responsive to noxious colonic stimulation. These data also indicate a change in the types of DH neurons activated by colonic mechanical stimuli post-inflammation. Such DH changes may correlate with increased pain perception associated with CVH. 1) Hughes & Brierley, et al. Gut. 2009.

Effects of Spinal Cord Section on Inspiratory Pressure Generation During High Frequency Spinal Cord Stimulation (HF‐SCS)

ObjectiveHF‐SCS is a novel and more physiologic method of inspiratory muscle activation which involves activation of spinal cord pathways. The specific pathways by which inspiratory motoneurons are activated by this method however are unknown. The aim of the present study was to evaluate the potential role of upper cervical inspiratory neurons on the observed responses.MethodsIn 6 anesthetized and spinalized (C1 level) dogs, HF‐SCS was applied at T2 level. Airway pressure generation (P) was monitored as an index of the degree of inspiratory muscle activation before and following sequential spinal sections at the C4 (to eliminate upper cervical propriospinal neurons) and subsequently the C8 level.ResultsDuring HF‐SCS, P at FRC was 68±6 cmH2O. Sequential bilateral dorsal columns (DC), lateral funiculi (LF) and complete section (CS) at C4, resulted in no significant changes in P (69±5, 67±5 and 65±7 cmH2O respectively, NS). While DC section at C8 resulted in no changes in P (63±5 cmH2O), LF section resulted in significant reduction in P to 18±3 cmH2O (p&lt;0.05). Subsequent CS resulted in no further reduction in P (14±1 cmH2O, NS).ConclusionDuring upper thoracic HF‐SCS, activation of the phrenic motoneuron pools does not involve upper cervical propriospinal inputs. The phrenic motoneuron pools are activated via spinal pathways located bilaterally in the lateral funiculi.Support: NIH‐NINDS (R01NS064157).

Intrathecal cannabilactone CB2R agonist, AM1710, controls pathological pain and restores basal cytokine levels

Spinal glial and proinflammatory cytokine actions are strongly implicated in pathological pain. Spinal administration of the anti-inflammatory cytokine interleukin (IL)-10 abolishes pathological pain and suppresses proinflammatory IL-1β and tumor necrosis factor alpha (TNF-α). Drugs that bind the cannabinoid type-2 receptor (CB2R) expressed on spinal glia reduce mechanical hypersensitivity. To better understand the CB2R-related anti-inflammatory profile of key anatomical nociceptive regions, we assessed mechanical hypersensitivity and protein profiles following intrathecal application of the cannabilactone CB2R agonist, AM1710, in 2 animal models; unilateral sciatic nerve chronic constriction injury (CCI), and spinal application of human immunodeficiency virus-1 glycoprotein 120 (gp120), a model of peri-spinal immune activation. In CCI animals, lumbar dorsal spinal cord and corresponding dorsal root ganglia (DRG) were evaluated by immunohistochemistry for expression of IL-10, IL-1β, phosphorylated p38-mitogen-activated-kinase (p-p38MAPK), a pathway associated with proinflammatory cytokine production, glial cell markers, and degradative endocannabinoid enzymes, including monoacylglycerol lipase (MAGL). AM1710 reversed bilateral mechanical hypersensitivity. CCI revealed decreased IL-10 expression in dorsal spinal cord and DRG, while AM1710 resulted in increased IL-10, comparable to controls. Adjacent DRG and spinal sections revealed increased IL-1β, p-p38MAPK, glial markers, and/or MAGL expression, while AM1710 suppressed all but spinal p-p38MAPK and microglial activation. In spinal gp120 animals, AM1710 prevented bilateral mechanical hypersensitivity. For comparison to immunohistochemistry, IL-1β and TNF-α protein quantification from lumbar spinal and DRG homogenates was determined, and revealed increased DRG IL-1β protein levels from gp120, that was robustly prevented by AM1710 pretreatment. Cannabilactone CB2R agonists are emerging as anti-inflammatory agents with pain therapeutic implications.

Musculotopic organization of the motor neurons supplying forelimb and shoulder girdle muscles in the mouse

We identified the motor neurons (MNs) supplying the shoulder girdle and forelimb muscles in the C57BL/6J mouse spinal cord using Fluoro-Gold retrograde tracer injections. In spinal cord transverse sections from C2 to T2, we observed two MN columns (medial and lateral) both with ventral and dorsal subdivisions. The dorsolateral column consisted of the biceps brachii, forearm extensors, forearm flexors, and hand MNs, and the ventrolateral column consisted of the latissimus dorsi, trapezius, teres major, deltoid, and triceps MNs. The supraspinatus muscle MNs were located in the dorsomedial column, and pectoralis major and serratus anterior MNs were located in the ventromedial columns. MNs of the dorsolateral column innervated the biceps brachii in mid-C4 to mid-C7, forearm extensors in caudal C4 to mid-T1, forearm flexors in rostral C5 to mid-T1, and hand muscles in mid-C8 to mid-T2 segments. The MNs innervating the trapezius were located in mid-C2 to mid-C4, triceps brachii in mid-C6 to rostral T1, deltoid in rostral C4 to mid-C6, teres major in rostral C5 to mid-C8, and latissimus dorsi in mid-C5 to caudal C8. In addition, MNs innervating the supraspinatus were located from rostral C4 to caudal C8, pectoralis major in mid-C6 to mid-T2, and serratus anterior in rostral C5 to caudal C7/rostral C8 segments. While the musculotopic pattern of MN groups was very similar to that documented for other species, we found differences in the position and cranio-caudal extent of some MN pools compared with previous reports. The identification of mouse forelimb MNs can serve as an anatomical reference for studying degenerative MN diseases, spinal cord injury, and developmental gene expression.

Vacuolation and mineralisation as dominant age-related findings in hamster brains

Syrian golden hamsters (Mesocricetus auratus) are laboratory animals increasingly used for research and toxicological studies. Despite the need for an adequate knowledge of spontaneously occurring lesions, studies investigating the background pathology of different organ systems in hamsters are lacking. The aim of this study was to investigate the occurrence of spontaneous, age-dependent lesions in the central nervous system of this species. Multiple brain and spinal cord transverse sections of 520 hamsters of 1, 3, 6, 12, and 24 months of age were investigated using histology and immunohistochemistry. Vacuolation of grey matter neuropil and mineralisation especially in the brain stem were the most prominent findings. They gradually increased in severity and frequency with age. Vacuolation and mineralisation affected approximately 100% and 50% of 24-month-old hamsters, respectively. In addition, pigment deposition and mast cell infiltration were commonly detected. Whether vacuolation and mineralisation represent an incidental finding or are related to a cognitive dysfunction syndrome remains to be determined.

Time course and mechanisms of motoneuron death in a type II spinal muscular atrophy mouse model

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease leading to motor impairment, muscle atrophy and premature death caused by motoneuron degeneration. It is caused by the deletion/mutation of the telomeric survival motoneuron gene (SMN1), whereas the number of copies of the centromeric gene SMN2, which produces reduced levels of functional protein, is inversely proportional to the severity of disease (from severe to mild). However, the causes of selective motoneuron death still remain elusive. To clarify the time course and the mechanisms of motoneuron (MN) death, we investigated the SMNdelta7 murine model of SMA II (the intermediate SMA form), in which motor dysfunction leads to death at P13. We collected brains and spinal cords from SMA II and wild type embryos/pups at E19, P4, P9 and P13 for neuron counts and immunohistochemistry. Newborns underwent a battery of motor tasks and were assessed daily for body weight and survival. In ChAT-immunoreacted and Nissl-stained spinal sections, stereological counts reported a dramatic reduction in the number of lower (cervical) MNs (almost 40% at P13) in the SMA II mice; in particular MNs innervating proximal muscles seemed the most affected. In addition, we noticed an increased ChAT expression through time, making ChAT-MN count less reliable than Nissl-ones. Moreover, even though most studies mainly report death of lower motoneurons, stereological counts in the motor cortex revealed a specific decrease of layer V cortical pyramidal neurons in SMA II mice compared to WT. Also the corpus callosum thickness appeared halved in the P9 SMA II mice. Finally, immunohistochemistry against cleaved Caspase-3 and LC-3 suggested an involvement of the apoptotic and autophagic modes of cell death, respectively. Therefore, at least in the animal model, SMA affects both upper and lower motoneurons, and SMN1 role in neuronal development and survival should be further investigated. Targeting apoptotic and autophagic pathways can delay the disease progression, as we are currently showing in other studies.

Lack of detectable diffuse or neuritic plaques and neurofibrillary tangles in the brains of aged hamsters

Syrian golden hamsters (Mesocricetus auratus) are facultative hibernators with a life expectancy of approximately 2 years. Previous investigations showed a hyperphosphorylation of the tau protein during hibernation and aging and raised hopes that Syrian hamsters might represent a useful animal model to study pathogenetic mechanisms of Alzheimer's disease. Brain and spinal cord transversal sections of 190 hamsters 1–36 months of age were investigated using histology and immunohistochemistry to detect neurofibrillary tangles and/or diffuse as well as neuritic plaques. Summarized, amyloid deposition, neurofibrillary tangles, and diffuse as well as neuritic plaques were absent indicating that the Syrian golden hamster does not develop changes characteristic of Alzheimer's disease even at advanced age and does not represent an appropriate animal model for this disease.

Exercise therapy and recovery after SCI: evidence that shows early intervention improves recovery of function

This was designed as an experimental study. Locomotor training is one of the most effective strategies currently available for facilitating recovery of function after an incomplete spinal cord injury (SCI). However, there is still controversy regarding the timing of treatment initiation for maximal recovery benefits. To address this issue, the present study compares the effects of exercise initiated in the acute and secondary phase of SCI. Texas A&M University, College Station, TX, USA. Rats received a moderate spinal contusion injury and began an exercise program 1 (D1-EX) or 8 days (D8-EX) later. They were individually placed into transparent exercise balls for 60 min per day, for 14 consecutive days. Control rats were placed in exercise balls that were rendered immobile. Motor and sensory recovery was assessed for 28 days after injury. The D1-EX rats recovered significantly more locomotor function (BBB scale) than controls and D8-EX rats. Moreover, analyses revealed that rats in the D8-EX group had significantly lower tactile reactivity thresholds compared with control and D1-EX rats, and symptoms of allodynia were not reversed by exercise. Rats in the D8-EX group also had significantly larger areas of damage across spinal sections caudal to the injury center compared with the D1-EX group. These results indicate that implementing an exercise regimen in the acute phase of SCI maximizes the potential for recovery of function.

Desorption Electrospray Ionization Imaging Mass Spectrometry of Lipids in Rat Spinal Cord

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The "butterfly" shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 mum was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.

Evidence of Central and Peripheral Sensitization in a Rat Model of Narcotic Bowel-Like Syndrome

Narcotic bowel syndrome (NBS) is a subset of opioid bowel dysfunctions that results from prolonged treatment with narcotics and is characterized by chronic abdominal pain. NBS is under-recognized and its molecular mechanisms are unknown. We aimed to (1) develop a rat model of NBS and (2) to investigate its peripheral and central neurobiological mechanisms. Male Wistar rats were given a slow-release emulsion that did or did not contain morphine (10 mg/kg) for 8 days. Visceral sensitivity to colorectal distension (CRD) was evaluated during and after multiple administrations of morphine or vehicle (controls). The effects of minocycline (a microglia inhibitor), nor-binaltorphimine (a kappa-opioid antagonist), and doxantrazole (a mast-cell inhibitor) were observed on morphine-induced visceral hyperalgesia. Levels of OX-42, P-p38 mitogen-activated protein kinase, rat mast cell protease II, and protein gene product 9.5 were assessed at different spinal segments (lumbar 6 to sacral 1) or colonic mucosa by immunohistochemistry. On day 8 of morphine administration, rats developed visceral hyperalgesia to CRD (incipient response) that lasted for 8 more days (delayed response). Minocycline reduced the incipient morphine-induced hypersensitivity response to CRD whereas nor-binaltorphimine and doxantrazole antagonized the delayed hyperalgesia. Levels of OX-42 and P-p38 increased in the spinal sections, whereas rat mast cell protease II and protein gene product 9.5 increased in the colonic mucosa of rats that were given morphine compared with controls. We developed a rat model of narcotic bowel-like syndrome and showed that spinal microglia activation mediates the development of morphine-induced visceral hyperalgesia; peripheral neuroimmune activation and spinal dynorphin release represent an important mechanism in the delayed and long-lasting morphine-induced colonic hypersensitivity response to CRD.

Sequestration of brain derived nerve factor by intravenous delivery of TrkB-Ig 2 reduces bladder overactivity and noxious input in animals with chronic cystitis

Brain derived nerve factor (BDNF) is a trophic factor belonging to the neurotrophin family. It is upregulated in various inflammatory conditions, where it may contribute to altered pain states. In cystitis, little is known about the relevance of BDNF in bladder-generated noxious input and bladder overactivity, a matter we investigated in the present study. Female rats were intraperitoneally (i.p.) injected with cyclophosphamide (CYP; 200 mg/kg). They received saline or TrkB-Ig 2 via intravenously (i.v.) or intravesical administration. Three days after CYP-injection, animals were anaesthetized and cystometries performed. All animals were perfusion-fixed and the spinal cord segments L6 collected, post-fixed and processed for c-Fos and phosphoERK immunoreactivity. BDNF expression in the bladder, as well as bladder histology, was also assessed. Intravesical TrkB-Ig 2 did not change bladder reflex activity of CYP-injected rats. In CYP-animals treated with i.v. TrkB-Ig 2 a decrease in the frequency of bladder reflex contractions, in comparison with saline-treated animals, was observed. In spinal sections from the latter group of animals, the number of phosphoERK and c-Fos immunoreactive neurons was lower than in sections from saline-treated CYP-animals. BDNF immunoreactivity was higher during cystitis but was not changed by TrkB-Ig 2 i.v. treatment. Evaluation of the bladder histology showed similar inflammatory signs in the bladders of inflamed animals, irrespective of the treatment. Data show that i.v. but not intravesical administration of TrkB-Ig 2 reduced bladder hyperactivity in animals with cystitis to levels comparable to those observed in unirritated rats. Since i.v. TrkB-Ig 2 also reduced spinal extracellular signal-regulated kinase (ERK) activation, it is possible that BDNF contribution to inflammation-induced bladder hyperactivity is via spinal activation of the ERK pathway. Finally, the reduction in c-Fos expression indicates that TrkB-Ig 2 also reduced bladder-generated noxious input. Our results show that sequestration of BDNF may be considered a new therapeutic strategy to treat chronic cystitis.

Effect of Huangqi On Mesenchymal Stem Cells (MSC) Transplanted Therapy in Spinal Cord Injury: It Promotes the Neural Differentiation of MSC In Vivo.

Abstract 4476 IntroductionThe therapeutic effects of using mesenchymal stem cells (MSCs) for transplantation in the treatment of spinal cord injury have been previously studied. Astragalus propinquus, also known as Huangqi, is a traditional Chinese herb commonly used to strengthen the immune system and protect neuron. However, the possibility of combining the beneficial effects of both MSC transplantation and Huangqi Injecta in treating spinal cord injury has not been addressed. MethodsA total of 120 male, four-week-old, Wistar rats, were randomly divided into six groups: sham group, model group, phosphate buffered saline (PBS) solution control group (PBS group), Huangqi injection control group (Huangqi group), rMSCs transplantation group (rMSCs group), rMSCs and Huangqi injecta treated group (rMSCs + Huangqi group). There were 10 rats in the sham group and 22 rats in the other groups. In the PBS group, rats were injected 5 μl PBS into the cephalic site of spinal cord injured section three days after the operation; in the Huangqi group, rats were intraperitoneally injected with Huangqi Injecta at a dose of 1.2 ml per 100 g of body weight on day three after the operation; in the rMSCs group, 5 μl of rMSC suspension (2.5–5×105 cells) was injected into the cephalic site of the spinal cord injured part in rats on day three after the operation; in the rMSCs + Huangqi group, rats were injected Huangqi Injecta and 5 μl rMSC suspension (2.5–5×105 cells) following the methods mentioned in the Huangqi group and the rMSCs group. Six animals were randomly picked from each groups on day 7, 14, 21 and 28 to receive Basso-Beattie-Bresnahan (BBB) scale grading for motor function checking of the hind legs. HE staining is adopt for the histopathologic examination, and Dual-marked immunohistochemical analysis is applied for cell locating and differentiation determination. ResultsThe average BBB score in the Huangqi group is higher than model group and PBS group, except on day 21. The score in rMSCs group and the rMSCs + Huangqi group is higher than that in the model group, PBS group and the Huangqi group. The rMSCs + Huangqi group has a higher score than the Huangqi group at each time point. Also, except on day 14, the score for the rMSCs + Huangqi group was higher than the rMSCs group, and the difference was significant. HE staining of the spinal cord paraffin sections showed that the degree of degeneration and necrosis in nerve cells was alleviated in the rMSCs and rMSCs + Huangqi groups, compared with the PBS and Huangqi groups. Edema and infiltration of inflammatory cells were obviously reduced, and the proliferation of glial cells and integrated nerve cells was detected in the rMSCs and rMSCs + Huangqi groups. Moreover, the proliferation of glial cells was more active in the rMSCs + Huangqi group than in the rMSCs group. Using immunohistochemical analysis, both in the rMSCs group and the rMSCs + Huangqi group, BrdU marked rMSCs survived in the spinal cord and accumulated in the gray matter more than in the white matter. GFAP + BrdU and BrdU+NF-M positive cells were detected from day 7 after transplantation. The quantity of BrdU + GFAP positive cells in the rMSCs group and rMSCs + Huangqi group is 6.35±1.14 and 8.75±1.16 per visual field relatively, while BrdU + NF-M positive cells in the rMSCs group and rMSCs + Huangqi group is 1.60±0.75 and 3.30±0.98 per visual field relatively. It indicated that the quantity of BrdU + GFAP and BrdU + NF-M positive cells per visual field observed in the spinal cord paraffin section of the rMSCs + Huangqi group, was significantly higher than that in the rMSCs group (P<0.05). ConclusionsThese findings indicated that MSCs transplantation has the capability of nerve restoration and recovery after spinal cord injury, and it could be enhanced by Huangqi Injecta. The strategy of combining Huangqi Injecta and MSCs, aimed to promote transplanted cell differentiation and their tissue repair capability, provided a simple yet effective way of optimizing cell transplantation therapy. Disclosures:No relevant conflicts of interest to declare.