Lumbar Enlargement Research Articles

Selective control of hindlimb movements based on intraspinal functional electronic stimulation

Functional electronic stimulation (FES) may provide a means to restore motor function in patients with spinal cord injuries. The goal of this study is to determine the regions in the spinal cord controlling different hindlimb movements in the rats. Normalization was used for the regions dominating the corresponding movements. It has been verified that FES can be used in motor function recovery of the hindlimb. The spinal cord was stimulated by FES with a three-dimensional scan mode in experiments. The results show that stimulation through the electrodes implanted in the ventral locations of the lumbosacral enlargement can produce coordinated single- and multi-joint hindlimb movements. A variety of different hindlimb movements can be induced with the appropriate stimulation sites, and movement vectors of the hindlimb cover the full range of movement directions in the sagittal plane of the hindlimb. This article drew a map about spinal cord motor function of the rat. The regions in the spinal cord which control corresponding movements are normalized. The data in the study provide guidance about the location of electrode tips in the follow-up experiments.

Effects of intrathecal injection Roscovitine on the pain behavior and the expression of ERK1/2 and PPARγ protein in spinal cord in rats with neuropathic pain

Objective To investigate the effects of intrathecal injection of Roscovitine on the pain behavior and the expression of pERK1/2 and pPPARγ receptor in spinal cord in rats with neuropathic pain. Methods Fifty-two male adult Sprague-Dawley rats of 220~250 g were randomly divided into 4 groups: Sham+ DMSO group (dimethyl sulfoxide, S+ D group), Sham+ Roscovitine (S+ R) group, CCI+ DMSO group (I+ D), CCI+ Roscovitine (I+ R) group. The corresponding drugs were administered by intrathecal injection from the seventh day after CCI once a day for 14 days. The right hind paw mechanical threshold value(PMWT) was measured by Von Frey filament and paw thermal withdrawal latency(PWTL) was measured by Hargreaves methods before 1 day and 3 d, 7 d, 10 d , 14 d after surgery. The spinal cord lumbar enlargement was taken at 14 d after surgery, and Cdk5, p35, phosphorylated ERK protein(pERK), total ERK protein(ERK), phosphorylated PPARγ protein (pPPARγ) and total PPARγ protein (PPARγ) were detecteded by Western blot. Results Roscovitine was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats.Compared with I+ D group, PWMT in I+ R group at 7 d, 10 d, 14 d after administration((4.65±0.08)g, ( 6.47±0.12)g, ( 7.90±0.19)g, ) vs ((3.71±0.06)g, (2.45±0.17)g, ( 2.31±0.15)g)(Fgroup=505.71, P<0.05, Ftime×group=15.33, P<0.05) , PWTL((9.22±0.33)s, (13.52±0.43)s, (12.63±0.88)s) vs ((8.02±0.20)s, (5.90±0.28)s, (5.40±0.38)s) (Fgroup=355, P<0.05, Ftime×group=8.589, P<0.05) were significantly increased. Compared with I+ D group(p35(0.58±0.02), pERK (1.12±0.13), pPPARγ (0.77±0.16)), p35 (0.46±0.04, F=11.06, P<0.05) and pERK(0.79±0.11, F=22.91, P<0.05) in I+ R group, were significantly dereased, and the expression of pPPARγ(0.99±0.13, F=17.62, P<0.05))was significantly increased. Conclusion Intrathecal injection Roscovitine can alleviate both mechanical allodynia and thermal hyperalgesia in rats with neuropathic pain, and its mechanisms may be related to the inhibition of Cdk5/p35 and ERK activity, enhancement of PPARγ activity in the spinal dorsal horn. Key words: Neuropathic pain; Cyclin-dependent protein kinases 5; Extracellular signal-regulated kinase1/2; Peroxisome proliferator-activated receptor gamma; Spinal dorsal horn

Intrathecal injection of AG-490 reduces bone-cancer-induced spinal cord astrocyte reaction and thermal hyperalgesia in a mouse model

To investigate the role of spinal interleukin-6-Janus kinase 2 (IL-6-JAK2) signaling transduction pathway in regulating astrocytes activation during the maintenance of bone cancer pain (BCP). Methods: NCTC 2472 fibrosarcoma cells were injected into the femur marrow cavity in C3H/HeNCrlVr male mice to establish BCP model and they were replaced by the equal volume of α-MEM in the sham model. The paw withdrawal latency (PWL) was measured after inoculation of tumor cells. The lumbar enlargement of spinal cord (L3-L5) was isolated, and Real-time RT-PCR and Western blot were used to detect the expression of spinal glial fibrillary acidic protein (GFAP) and JAK2 mRNA and protein, respectively. The expression level of spinal GFAP mRNA indirectly reflect astrocytes activation level. Pain behaviors and spinal cord GFAP mRNA and protein expression were observed at the given time points after intrathecal administration of JAK2 antagonist AG-490. Results: The PWL at 10, 14, 21 d after operation in BCP model group were significantly shorter than that in the sham group (P<0.05); the spinal GFAP and JAK2 mRNA and protein levels were higher in the BCP model group in comparison to mice in the sham group (P<0.05); intrathecal injection of JAK2 agonist AG-490 (30 or 90 nmol) significantly alleviated PWL, and downregulated the expression of spinal GFAP mRNA and protein (P<0.05). Conclusion: The IL-6-JAK2 signaling pathway plays an important role in maintaining the BCP by regulating the expression of GFAP in the spinal cord. Intrathecal injection of AG-490 can reduce the BCP, and inhibit the activation of IL-6-JAK2 signaling pathway, which may be one of the mechanisms for spinal astrocyte activation.

A speed-adaptive intraspinal microstimulation controller to restore weight-bearing stepping in a spinal cord hemisection model

Objective. The goal of this study was to develop control strategies to produce alternating, weight-bearing stepping in a cat model of hemisection spinal cord injury (SCI) using intraspinal microstimulation (ISMS). Approach. Six cats were anesthetized and the functional consequences of a hemisection SCI were simulated by manually moving one hind-limb through the gait cycle over a moving treadmill belt. ISMS activated the muscles in the other leg by stimulating motor networks in the lumbosacral enlargement using low levels of current (<110 µA). The control strategy used signals from ground reaction forces and angular velocity from the manually-moved limb to anticipate states of the gait cycle, and controlled ISMS to move the other hind-limb into the opposite state. Adaptive control strategies were developed to ensure weight-bearing at different stepping speeds. The step period was predicted using generalizations obtained through four supervised machine learning algorithms and used to adapt the control strategy for faster steps. Main results. At a single speed, 100% of the steps had sufficient weight-bearing; at faster speeds without adaptation, 97.6% of steps were weight-bearing (significantly less than that for single speed; p = 0.002). By adapting the control strategy for faster steps using the predicted step period, weight-bearing was achieved in more than 99% of the steps in three of four methods (significantly more than without adaptation p < 0.04). Overall, a multivariate model tree increased the number of weight-bearing steps, restored step symmetry, and maintained alternation at faster stepping speeds. Significance. Through the adaptive control strategies guided by supervised machine learning, we were able to restore weight-bearing and maintain alternation and step symmetry at varying stepping speeds.

Retrogradely Transportable Lentivirus Tracers for Mapping Spinal Cord Locomotor Circuits.

Retrograde tracing is a key facet of neuroanatomical studies involving long distance projection neurons. Previous groups have utilized a variety of tools ranging from classical chemical tracers to newer methods employing viruses for gene delivery. Here, we highlight the usage of a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating motor neuron pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. Our study demonstrates that the HiRet lentivirus is a unique tool for examining neuronal circuitry within the brain and spinal cord.

The Amelioration of Pain-Related Behavior in Mice with Chronic Spinal Cord Injury Treated with Neural Stem/Progenitor Cell Transplantation Combined with Treadmill Training.

Progress in regenerative medicine is realizing the possibility of neural regeneration and functional recovery in spinal cord injury (SCI). Recently, rehabilitation has attracted much attention with respect to the synergistic promotion of functional recovery in combination with neural stem/progenitor cell (NS/PC) transplantation, even in the chronic refractory phase of SCI. Nevertheless, sensory disturbance is one of the most prominent sequelae, even though the effects of combination or single therapies have been investigated mostly in the context of motor recovery. To determine how combination therapy with treadmill training (TMT) and NS/PC transplantation affects the manifestation of thermal allodynia and tactile hyperalgesia in chronic phase SCI, four groups of SCI mice were used to assess pain-related behavior and histological changes: combined transplantation and TMT therapy, transplantation only, TMT only, and control groups. Thermal allodynia and coarse touch-pressure hyperalgesia exhibited significant recovery in the combined therapy group in comparison with controls, whereas there were no significant differences with fine touch-pressure hyperalgesia and motor function. Further investigation revealed fewer fibers remaining in the posterior funiculus, which contained the tracts associated with the two modalities showing less recovery; that is, touch-pressure hyperalgesia and motor function. A significant correlation was only observed between these two modalities. Although no remarkable histological recovery was found within the lesion epicenter, changes indicating amelioration of pain were observed in the lumbar enlargement of the combination therapy group. Our results suggest that amelioration of thermal allodynia and tactile hyperalgesia can be brought about by the additive effect of NS/PC transplantation and TMT. The degree of recovery seems dependent on the distribution of damage.

Effect of exogenous resolvin D2 on radicular pain in a rat model of non-compressive lumbar intervertebral disc herniation

Objective To evaluate the effect of exogenous resolvin D2 on radicular pain in a rat model of non-compressive lumbar intervertebral disc herniation. Methods Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 230-270 g, were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), radicular pain induced by non-compressive lumbar intervertebral disc herniation group (group P) and exogenous resolvin D2 group (group R). The right L5 dorsal root ganglions were covered by autologous nucleus pulposus tissues to establish the model of non-compressive lumbar disc herniation in P and R groups.The corresponding surgical site was only exposed in group S. The corresponding drugs were intrathecally injected within 3 days after establishing the model, phosphate buffer solution 10 μl was injected in group P, 1 ng/μl resolvin D2 solution 10 μl was injected in group R, and normal saline 10 μl was given for pipe washing after administration in the three groups.The mechanical paw withdrawal threshold (MWT) was measured on 1 day before establishing the model and 1-7 days after establishing the model.The spinal dorsal horns of lumbar enlargement segments were removed on day 7 after establishing the model for determination of the expression of glial fibrillary acidic protein (GFAP) (by Western blot) and co-expression of G-protein-coupled receptor 18 with GFAP (by double-label immunofluorescence). Results Compared with group S, the MWT was significantly decreased at 1-7 days after establishing the model, and the expression of GFAP was up-regulated in group P (P<0.05). Compared with group P, the MWT was significantly increased at 3-7 days after establishing the model, and the expression of GFAP was down-regulated in group R (P<0.05). G-protein-coupled receptor 18 was co-expressed with GFAP. Conclusion Exogenous resolvin D2 can reduce radicular pain in a rat model of non-compressive lumbar intervertebral disc herniation, and the mechanism is related to inhibiting activation of astrocytes in the spinal dorsal horns. Key words: Docosahexaenoic acids; Neuralgia; Intervertebral disc displacement; Glial fibrillary acidic protein

Constitutively reduced sensory capacity promotes better recovery after spinal cord-injury (SCI) in blind rats of the dystrophic RCS strain

We compared functional, electrophysiological and morphological parameters after SCI in two groups of rats Sprague Dawley (SD) rats with normal vision and blind rats from a SD-substrain "Royal College of Surgeons" (SD/RCS) who lose their photoreceptor cells after birth due to a genetic defect in the retinal pigment epithelium. For these animals skin-, intramuscular-, and tendon receptors are major available means to resolve spatial information. The purpose of this study was to check whether increased sensitivity in SD/RCS rats would promote an improved recovery after SCI. All rats were subjected to severe compression of the spinal cord at vertebra Th8, spinal cord segment Th10. Recovery of locomotion was analyzed at 1, 3, 6, 9, and 12 weeks after SCI using video recordings of beam walking and inclined ladder climbing. Five functional parameters were studied: foot-stepping angle (FSA), rump-height index (RHI) estimating paw placement and body weight support, respectively, number of correct ladder steps (CLS) assessing skilled hindlimb movements, the BBB-locomotor score and an established urinary bladder score (BS). Sensitivity tests were followed by electrophysiological measurement of M- and H-wave amplitudes from contractions of the plantar musculature after stimulation of the tibial nerve. The closing morphological measurements included lesion volume and expression of astro- and microglia below the lesion. Numerical assessments of BBB, FSA, BS, lesion volume and GFAP-expression revealed no significant differences between both strains. However, compared to SD-rats, the blind SD/RCS animals significantly improved RHI and CLS by 6 - 12 weeks after SCI. To our surprise the withdrawal latencies in the blind SD/RCS rats were longer and the amplitudes of M- and H-waves lower. The expression of IBA1-immunoreactivity in the lumbar enlargement was lower than in the SD-animals. The longer withdrawal latencies suggest a decreased sensitivity in the blind SD/RCS rats, which promotes better recovery after SCI. In this way our results provide indirect support to earlier work showing, that hypersensitivity and chronic pain after contusive SCI impair the recovery of locomotor function.

Changes in expression of Piezo2 in spinal cord neurons in a rat model of bone cancer pain

Objective To evaluate the changes in the expression of Piezo2 in spinal cord neurons in a rat model of bone cancer pain. Methods Sixty-four pathogen-free healthy adult female unmated Sprague-Dawley rats, weighing 160-200 g, were divided into 2 groups (n=32 each) using a random number table: sham operation group (group S) and bone cancer pain group (group BP). Bone cancer pain was induced by injecting breast cancer cells into the abdominal cavity.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before inoculating breast cancer cells and 7, 14 and 21 days after inoculation (T0-3). Eight rats were sacrificed after measurement of MWT, and their lumbar enlargement segments of the spinal cord were removed for determination of Piezo2 expression by Western blot and immunofluorescence.The coexpression of Piezo2 with the neuronal marker NeuN, microglial marker Iba-1 and astrocyte marker GFAP was detected at T2 using double immunofluorescent staining. Results Compared with group S, the MWT was significantly decreased at T1-3, and the Piezo2 expression in the lumbar enlargement segment of the spinal cord was up-regulated in group BP (P < 0.05). Piezo2 was mainly expressed in the spinal lamina Ⅰ and Ⅱ and co-expressed with NeuN and rarely co-expressed with GFAP or Iba-1. Conclusion The development and maintenance of bone cancer pain are related to up-regulated expression of Piezo2 in the lumbar enlargement segment of the spinal cord in rats. Key words: Bone neoplasms; Pain; Spinal cord; Neuron; Piezo2

Isoflurane anesthesia does not affect spinal cord neurovascular coupling: evidence from decerebrated rats.

Neurological examination remains the primary clinical investigation in patients with spinal cord injury. However, neuroimaging methods such as functional magnetic resonance imaging (fMRI) are promising tools for following functional changes in the course of injury, disease and rehabilitation. However, the relationship between neuronal activity and blood flow in the spinal cord on which fMRI relies has been largely overlooked. The objective of this study was to examine neurovascular coupling in the spinal cord of decerebrated rats during electrical stimulation of the sciatic nerve with and without isoflurane anesthesia (1.2%). Local field potentials (LFP) and spinal cord blood flow (SCBF) were recorded simultaneously in the lumbosacral enlargement. Isoflurane did not significantly alter LFP (p = 0.53) and SCBF (p = 0.57) amplitude. Accordingly, neurovascular coupling remained comparable with or without isoflurane anesthesia (p = 0.39). These results support the use of isoflurane in rodents to investigate nociceptive functions of the spinal cord using fMRI.

Substance P-expressing excitatory interneurons in the mouse superficial dorsal horn provide a propriospinal input to the lateral spinal nucleus

The superficial dorsal horn (laminae I and II) of the spinal cord contains numerous excitatory and inhibitory interneurons, and recent studies have shown that each of these groups can be divided into several neurochemically distinct populations. Although it has long been known that some neurons in this region have intersegmental (propriospinal) axonal projections, there have been conflicting reports concerning the number of propriospinal cells and the extent of their axons. In addition, little is known about the neurochemical phenotype of propriospinal neurons or about the termination pattern of their axons. In the present study we show, using retrograde tracing, that around a third of lamina I–II neurons in the lumbar enlargement project at least five segments cranially. Substance P-expressing excitatory neurons are over-represented among these cells, accounting for one-third of the propriospinal neurons. In contrast, inhibitory interneurons and excitatory PKCγ neurons are both under-represented among the retrogradely labelled cells. By combining viral vector-mediated Cre-dependent anterograde tracing with immunocytochemistry, we provide evidence that the lateral spinal nucleus (LSN), rather than the superficial dorsal horn, is the main target for axons belonging to propriospinal substance P-expressing neurons. These findings help to resolve the discrepancies between earlier studies and have implications for the role of the LSN in pain mechanisms.

Immunohistochemical Expression of Synaptophysin in the Adult and Developing Cervical and Lumbar Enlargements of the Spinal Cord of Rabbit

Background: The principle findings of synaptophysin immunoreactivity (SynpIR) during the ontogeny of rabbit spinal cord are: At E14, SynpIR precedes in the entire marginal layer especially at the entrance zone of dorsal root and motor neurite outgrowth emerged from the basal plate. At E21, SynpIR is expressed in the motoneurons of ventral and lateral horns of mantle layer growing into the ventrolateral columns of marginal layer. Methods: We found intensely stained thick tracts and diffuse axons among proliferating neuroblasts of mantle layer. The peripheral parts of ventral horns were occupied with closely packed multipolar neurons from which long dendrites departed toward the surface of marginal layer. Results: At E28, pronounced SynpIR presented in the ventral grey horn while the white matter was faintly stained., meanwhile the dorsal horn was more cellular than ventral and lateral horns. Few intensively SynpIR fibers cross the dorsal and ventral commissures. In adult, profuse SynpIR appeared in the entire grey matter, and stained dendrites departed from neurons in the lateral laminae into the adjacent funiculi as finger-like projections. These projections did not reach the surface, so that the outer one-third to onefourth of the funiculi contained little or no SynpIR. In the periphery of ventral horns, we found large multipolar neurons with faintly stained cytoplasm. The white matter and the neuroepithelial cells surrounding the central canal were almost unstained. Conclusion: Synaptophysinis a reliable marker for fiber outgrowth and synapse formation in therabbit spinal cord, and its differential expression levels is specific and almost completed before birth.

Changes in expression of spinal endothelin-1 and its receptors in a mouse model of bone cancer pain

Objective To evaluate the changes in the expression of spinal endothelin-1(ET-1)and its receptors in a mouse model of bone cancer pain(BCP). Methods Ninety-six healthy male SPF C3H/HeN mice, aged 4-6 weeks, weighing 20-25 g, were divided into 2 groups(n=48 each)using a random number table: sham operation group(group S)and BCP group.BCP was produced by injecting α-MEM 20 μl containing 1×104 cells/μl NCTC 2472 osteosarcoma cells into the distal medullary cavity of the right femur bone.In group S, α-MEM 20 μl was injected into the distal medullary cavity of the right femur bone.Mechanical paw withdrawal threshold(MWT)and the number of spontaneous flinches(NSF)were measured on 1 day before inoculation(T0)and 4, 7, 10, 14 and 21 days after inoculation(T1-5). Twelve mice of each group were randomly sacrificed at T0, 2, 4, 5, and the lumbar enlargement segments of the spinal cord were harvested to detect the expression of ET-1, endothelin type A receptor and endothelin type B receptor protein and mRNA(using Western blot or real-time polymerase chain reaction). Results The MWT was significantly lower and the NSF was higher at T1 in group S and at T1-5 in group BCP than at T0(P<0.05). Compared with group S, the MWT was significantly decreased and the NSF was increased at T2-5, and the expression of ET-1, endothelin type A receptor and endothelin type B receptor protein and mRNA was down-regulated at T2, 4, 5 in group BCP(P<0.05). Conclusion The pathophysiological process of BCP is associated with down-regulating the expression of spinal ET-1 and its receptors in mice. Key words: Receptor, endothelin type A; Receptor, endothelin type B; Receptors, G protein-coupled; Bone neoplasms; Pain; Spinal cord

Role of caspase-1 in spinal cord in a rat model of incisional pain

Objective To evaluate the role of caspase-1 in the spinal cord in a rat model of incisional pain. Methods Eighteen adult male Sprague-Dawley rats, weighing 250-280 g, in which intrathecal catheters were successfully implanted, were divided into 3 groups(n=6 each)using a random number table: incision pain group(group I), incision pain plus dimethyl sulfoxide group(group ID)and incision pain plus caspase-1 inhibitor(Ac-YVAD-CMK)group(group IA). At 10 min before establishment of the model, 0.9% normal saline 20 μl was intrathecally injected in group I, dimethyl sulfoxide 20 μl was intrathecally injected and then the catheter was washed with 0.9% normal saline 10 μl in group ID, and Ac-YVAD-CMK 1 nmol/μl(dissolved in 20 μl dimethyl sulfoxide)and then the catheter was washed with 0.9% normal saline 10 μl in group IA.The mechanical paw withdrawal threshold(MWT)of the ipsilateral hind paw was measured at 2 h before intrathecal catheterization(T0), 3 days after intrathecal catheterization(T1)and 2, 6, 24 and 48 h after establishment of model(T2-5). The rats were sacrificed after the last measurement of pain threshold at T5, and lumbar enlargement segments of the spinal cord were removed for detection of caspase-1(p20)expression and interleukin-1beta(IL-1β)content by Western blot and enzyme-linked immunosorbent assay, respectively. Results Compared with the baseline at T0, the MWT was significantly decreased at T2-5 in I and ID groups(P 0.05). Compared with I and ID groups, the MWT at T2-5 was significantly increased at T2-5, and the caspase-1(p20)protein expression and IL-1β content were decreased in group IA(P 0.05). Conclusion The activation of caspase-1 in the spinal cord can promote the release of IL-1β and thus is involved in the incision pain in rats. Key words: Pain, postoperative; Caspase1; Interleukin-1beta; Spinal cord

Minocycline attenuates the development of diabetic neuropathy by inhibiting spinal cord Notch signaling in rat

We studied the effects of minocycline (an inhibitor of microglial activation) on the expression and activity of Notch-1 receptor, and explored the therapeutic efficacy of minocycline combined with Notch inhibitor DAPT in the treatment of diabetic neuropathic pain (DNP). Diabetic rat model was established by intraperitoneal injection (ip) of Streptozotocin (STZ). Expression and activity of Notch-1 and expression of macrophage/microglia marker Iba-1 were detected by WB. Diabetes induction significantly attenuated sciatic nerve conduction velocity, and dramatically augmented the expression and the activity of Notch-1 in the lumbar enlargement of the spinal cord. Minocycline treatment, however, accelerated the decreased conduction velocity of sciatic nerve and suppressed Notch-1expression and activity in diabetic rats. Similar to DAPT treatment, minocycline administration also prolonged thermal withdrawal latency (TWL) and increase mechanical withdrawal threshold (MWT) in diabetic rats in response to heat or mechanical stimulation via inhibition the expression and the activity of Notch-1 in spinal cord. Combination of DAPT and minocycline further inhibited Notch-1 receptor signaling and reduce neuropathic pain exhibited as improved TWL and MWT. Our study revealed a novel mechanism of Notch-1 receptor inhibition in spinal cord induced by minocycline administration, and suggested that the combination of minocycline and DAPT has the potential to treat DNP.

Chronic constriction injury of sciatic nerve changes circular RNA expression in rat spinal dorsal horn.

BackgroundMechanisms of neuropathic pain are still largely unknown. Molecular changes in spinal dorsal horn may contribute to the initiation and development of neuropathic pain. Circular RNAs (circRNAs) have been identified as microRNA sponges and involved in various biological processes, but whether their expression profile changes in neuropathic pain condition is not reported.MethodsTo test whether neuropathic pain influences circRNA expression, we developed a sciatic chronic constriction injury (CCI) model in rats. The CCI ipsilateral spinal dorsal horns of lumbar enlargement segments (L3–L5) were collected, and the total RNA was extracted and subjected to Arraystar Rat circRNA Microarray. Quantitative real-time polymerase chain reaction (qPCR) was used to confirm the circRNA expression profile. To estimate functions of differential circRNAs, bioinformatics analyses including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes Pathway analyses were performed for the top 100 circRNAs and circRNA–microRNA networks were constructed for the top 10 circRNAs.ResultscircRNA microarrays showed that 469 circRNAs were differentially expressed between CCI and sham-operated rats (fold change ≥2). In all, 363 of them were significantly upregulated, and the other 106 were downregulated in the CCI group. Three of them (circRNA_013779, circRNA_008008, and circRNA_003724) overexpressed >10 times after CCI insult. Expression levels of eight circRNAs were verified using qPCR. GO analysis revealed that thousands of predicted target genes were involved in the biological processes, cellular component, and molecular function; in addition, dozens of these genes were enriched in the Hippo signaling pathway, MAPK signaling pathway, and so on. Competing endogenous RNAs analysis showed that circRNA_008008 and circRNA_013779 are the two largest nodes in the circRNA–microRNA interaction network of the top 10 circRNAs.ConclusionCCI resulted in a comprehensive expression profile of circRNAs in the spinal dorsal horn in rats. CircRNAs in the dorsal horn could be helpful to reveal molecular mechanisms of neuropathic pain.

Ulinastatin attenuates neuropathic pain via the ATP/P2Y2 receptor pathway in rat models

Ulinastatin, a serine protease inhibitor, which has anti-inflammatory properties and neuroprotective effects, is used to treat acute inflammatory disorders. Recent evidence indicates that administration of ulinastatin alleviates pain in rat model of neuropathic pain (NPP). However, its effect on NPP and the underlying mechanism requires further study. In this study, we evaluated the role of intrathecal administration of ulinastatin in rats with sciatic nerve ligation and observed the effect of ulinastatin on the ATP/P2Y2 receptor pathway. We performed mechanical and thermal sensitivity measurements, immunohistochemistry and double-label immunofluoresence studies to evaluate P2Y2 receptor and adenosine 5′-monophosphate-activated protein kinase (AMPK) expression in the dorsal horn of the lumbar enlargement region of the spinal cord, and a luciferase assay for the detection of ATP levels in the cerebrospinal fluid. The results showed that ulinastatin prevented the development of mechanical allodynia and thermal hypersensitivity in the rat sciatic nerve ligation model. Ulinastatin reduced the level of extracellular ATP, down-regulated P2Y2 receptor and AMPK expression in the spinal dorsal horn of the chronic constrictive injury model. We found that increased expression of P2Y2 receptor in microglia was likely involved in the activation of microglia after nerve injury, and ulinastatin inhibited the abnormal microglia activation in the dorsal horn after nerve injury. These findings demonstrated that ulinastatin might be a potential and effective drug for the treatment of NPP via the suppression of the ATP/P2Y2 receptor pathway.

A novel approach for assigning levels to monkey and human lumbosacral spinal cord based on ventral horn morphology.

Proper identification of spinal cord levels is crucial for clinical-pathological and imaging studies in humans, but can be a challenge given technical limitations. We have previously demonstrated in non-primate models that the contours of the spinal ventral horn are determined by the position of motoneuron pools. These positions are preserved within and among individuals and can be used to identify lumbosacral spinal levels. Here we tested the hypothesis that this approach can be extended to identify monkey and human spinal levels. In 7 rhesus monkeys, we retrogradely labeled motoneuron pools that represent rostral, middle and caudal landmarks of the lumbosacral enlargement. We then aligned the lumbosacral enlargements among animals using absolute length, segmental level or a relative scale based upon rostral and caudal landmarks. Inter-animal matching of labeled motoneurons across the lumbosacral enlargement was most precise when using internal landmarks. We then reconstructed 3 human lumbosacral spinal cords, and aligned these based upon homologous internal landmarks. Changes in shape of the ventral horn were consistent among human subjects using this relative scale, despite marked differences in absolute length or age. These data suggest that the relative position of spinal motoneuron pools is conserved across species, including primates. Therefore, in clinical-pathological or imaging studies in humans, one can assign spinal cord levels to even single sections by matching ventral horn shape to standardized series.

Tight neurovascular coupling in the spinal cord during nociceptive stimulation in intact and spinal rats

Functional magnetic resonance imaging (fMRI) is based on neurovascular coupling, which allows inferring neuronal activity from hemodynamic changes. Spinal fMRI has been used to examine pain processes, although spinal neurovascular coupling has never been investigated. In addition, fluctuations in mean arterial pressure (MAP) occur during nociceptive stimulation and this may affect neurovascular coupling. The objective of this study was to examine neurovascular coupling in the rat spinal cord during nociceptive stimulation while MAP was manipulated by cervical spinal transection, which prevents nociception-related MAP increases. Six male Wistar rats were anesthetized with isoflurane (1.2–1.5%). Local field potentials (LFP) and spinal cord blood flow (SCBF) were recorded concurrently in the lumbar enlargement, where activity was evoked by electrical stimulation of the sciatic nerve. In intact conditions, stimulation of graded intensity produced proportional changes in SCBF and LFP that were paralleled by similar changes in MAP. However, spinal transection almost abolished MAP changes (p<0.001), while SCBF and LFP responses were not significantly affected (p>0.3) and remained similarly coupled before and after spinal transection. This indicates that spinal hemodynamic changes reflect neuronal activity even when large fluctuations in MAP occur. This contrasts with results from previous studies on cerebral neurovascular coupling and suggests that spinal autoregulation might allow better adaptation to sudden MAP changes than cerebral autoregulation. Although assessment of the coupling between spinal neuronal activity and BOLD signal remains to be investigated, this study supports the use of spinal fMRI, based on the tight coupling between SCBF and LFP.

Peculiarities of spinal cord segments structural organization in human fetuses with anencephaly of 17–18 weeks of intrauterine development

The principles of the human spinal cord development constitute the basis for understanding of appearance of congenital abnormalities of the CNS development, which make 7 % of the total number of malformations in the embryonic period of human’s development.Purpose – to find out the cytoarchitectonic structure of the spinal cord segments of human fetuses with anencephaly, their morphometric parameters and the degree of the proliferation of neural stem cells.Materials and methods – anatomical and histological, immunohistochemical and morphometric research was carried out related to the spinal cord of 4 human fetuses with anencephaly of the gestational period of 17–18 weeks.Results. The length of the spinal cord of 17–18 weeks anencephalic fetuses corresponds to the length of human fetuses without malformations of 11–12 weeks, the rest macrometric parameters – 8–9 weeks. The largest area of grey matter was that of segments at the level of lumbosacral and cervical enlargements, the least one at the level of thoracic segments. The specific gravity of neuroepithelium in the grey matter of cervical, thoracic, lumbar and sacral segments was 0.4, 0.1, 0.6, 1.6 % respectively. The expression of the proliferation protein in the spinal cord neuroepithelium segments of anencephalic fetuses is approaching 0 %. However, in the ventral horns near the location of neural complexes single mitoses of glial cells occur. In quantitative terms, in the ventral horns 1.5 % of glial cells reacted, while in the dorsal ones – 0.6 %. The expression of vimentin in the mantle and marginal layers is poor. On the border between the ventral and dorsal horns an insignificant number of fibers have a radial direction. Moderate expression of synaptophysin was observed in the marginal layer, except for cuneate fasciculi where the relatively weak expression has been established, which testifies to the insufficient myelination of the conduction tracts fibers.Conclusions. Macro- and morphometric parameters of the spinal cord of human fetuses with 17–18 week anencephaly point out to the significant retardation in development. Numerous disorders in the structural organization of both grey matter and white matter have been found out. The proliferation of neural stem cells in neuroepithelium is approaching 0 %.