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Resumen Introducción. Es escasa la información sobre los detalles neuroanatómicos del transporte del virus de la rabia en su ascenso por la médula espinal. Objetivos. Identificar la ruta neuroanatómica de diseminación del virus de la rabia en cada uno de los niveles de la médula espinal de ratón, después de ser inoculado por vía intramuscular. Materiales y métodos. Se inocularon ratones en los músculos isquiotibiales, con virus de la rabia. A partir de las 24 horas después de la inoculación, cada ocho horas se sacrificaron cinco animales por perfusión con paraformaldehído, se les extrajo la médula espinal y se hicieron cortes transversales en los niveles lumbosacro, torácico y cervical. Estos se procesaron mediante inmunohistoquímica para detectar antígenos virales. Resultados. Los primeros antígenos de la rabia se observaron como partículas agregadas, en la médula espinal lumbar, a las 24 horas después de la inoculación, dentro del asta ventral ipsilateral a la extremidad inoculada. A las 32 horas después de la inoculación, se hicieron visibles las primeras motoneuronas inmunorreactivas al virus. A las 40 horas después de la inoculación, se revelaron las primeras neuronas inmunorreactivas en la médula torácica, localizadas en la lámina 8 y, a las 48 horas después de la inoculación en la médula cervical, también en la lámina 8. A las 56 horas después de la inoculación, el virus se había diseminado por toda la médula espinal pero los animales aún no revelaban signos de la enfermedad. Conclusión. En el modelo de ratón aquí utilizado, el virus de la rabia ingresó a la médula espinal por las motoneuronas y, probablemente, utilizó la vía propioespinal descendente para su transporte axonal retrógrado hasta el encéfalo.
Abstract Introduction: Information about the neuroanatomical details of the ascendant transport of the rabies virus through the spinal cord is scarce. Objective: To identify the neuroanatomical route of dissemination of the rabies virus at each of the levels of the spinal cord of mice after being inoculated intramuscularly. Materials and methods: Mice were inoculated with the rabies virus in the hamstrings. After 24 hours post-inoculation, every eight hours, five animals were sacrificed by perfusion with paraformaldehyde. Then, the spinal cord was removed, and transverse cuts were made at the lumbosacral, thoracic, and cervical levels. These were processed by immunohistochemistry for the detection of viral antigens. Results: The first antigens of rabies were observed as aggregated particles in the lumbar spinal cord at 24 hours post-inoculation, within the ventral horn in the same side of the inoculated limb. At 32 hours post inoculation the first motoneurons immunoreactive to the virus became visible. At 40 hours post-inoculation the first immunoreactive neurons were revealed in the thoracic level, located on lamina 8 and at 48 hours post-inoculation in the cervical cord, also on lamina 8. At 56 hours post-inoculation the virus had spread throughout the spinal cord, but the animals still did not show signs of the disease. Conclusion: In the mouse model we used, the rabies virus entered the spinal cord through the motoneurons and probably used the descending propriospinal pathway for its retrograde axonal transport to the encephalus.
Subject(s)
Animals , Female , Mice , Rabies virus/physiology , Spinal Cord/virology , Spinal Cord/anatomy & histologyABSTRACT
Objective To explore the analgesic effect of intra-articular botulinum neurotoxin type A (BoNTA) injection in rats with adjuvant-arthritis pain,to quantify the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglia (DRG) associated with arthritis pain,and to investigate the retrograde axonal transport of BoNT-A into the DRG after peripheral injection.Methods Ninety Sprague-Dawley rats were randomly divided into groups A,B,C,D and E,each of 18.A murine model of adjuvant-arthritis pain was established by injecting 50 μL of complete Freund's adjuvant into the left ankle in all the mice except those in group A.The control group A was treated with intra-articular injection of 50 μL of saline solution.Three weeks later,groups A and B were treated with a 20 μL intra-articular saline injection,while groups C,D and E received an intra-articular injection of BoNT-A at 1 U/20 μL,3 U/20 μL or 10 U/20 μL respectively.Pain threshold and muscle strength were graded before and 1,5,15 and 21 days after the modelling,as well as at 1,3,5 and 14 days after the BoNT-A treatments.Protein expression and the CGRP-positive cell number were observed,as well as any BoNT-A-cleaved synaptosomal-associated 25 kDa protein (cl-SNAP-25) in the DRG using Western blotting and immunofluorescence.Results Compared with group A,there was a significant decrease in the average mechanical withdrawal threshold and muscle strength and a significant increase in the protein expression and the CGRP-positive cell number in the other 4 groups.Compared with group B,the mechanical withdrawal threshold had increased significantly more in groups D and E at 5 days after the BoNT-A injection and in group C at 14 days after the treatment.Compared with group B,the protein expression and the number of CGRP-positive cells were significantly lower in groups D and E at 3 days after the BoNT-A injection.The decrease in group C was significant after 14 days.No significant differences were found between groups D and E in any measurement at any time point.There was no significant difference among groups B,C and D in terms of muscle strength.Five days after the BoNT-A injection,significantly decreased muscle strength was observed in group E.In addition,BoNT-A cleaved-SNAP-25 was detected in the DRG.Conclusion BoNT-A can reduce arthritis pain through inhibiting the expression of CGRP in the DRG.Its analgesic effect has a dose response.A peripheral injection of BoNT-A can arrive at the DRG through retrograde axonal transport.
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Objective To investigate the effect of tourniquet compression on axonal transport in sciatic nerve of rats.Methods Twenty-four 12-week old male SD rats weighing 250-300 g were randomly divided into 4groups according to the duration of tourniquet compression(n=6 each):1,2,4 and 12 h.The tourniquet was applied to the middle 1/3 of thigh.In each animal whether the left or right thigh was compressed was determined by a flip of coin.The tourniquet was released for 10 min after every hour of compression.A 3-cm segment of sciatic nerve was removed at the end of tourniquet compression(1.5 cm proximal and 1.5 cm distal to the site of compression).Immuno-histochemistry was used to measure the expression of insulin-like growth factor-1(IGF-1)in the sciatic nerve.The ratio of average optic density of the compressed sciatic nerve to that of control was used to estimate the degree of IGF-1 accumulation.The regression equation of the interaction between the duration of compression and accumulation of IGF-1 was analyzed.Results There was significant accumulation of IGF-1 in the sciatic nerve proximal to the compressed site.The accumulation increased with the duration of compression.There was no significant accumulation of IGF-1 in the sciatic nerve distal to the compressed site.The regression equation of the interaction between the duration of compression(X)and accumulation of IGF-1(Y)was Y=0.422X+0.887.Conclusion Tourniquet compression of sciatic nerve can inhibit axonal transport.The accumulation increases with the duration of compression.
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PURPOSE: To investigate the replication of HSV within cultured cell and axonal transport of HSV within the axon of the ciliary nerve following the injection of HSV into a cultured ciliary nerve. METHODS: The explant of the ciliary nerve was cultured with a medium containing nerve growth factor for 30 days when the suspension of HSV-1 (Kos strain) was introduced into the culture dish to co-culture with the ciliary nerve. The ciliary nerve was examined with transmission electron microscopy 30 days after culture and 6 days after co-culture with HSV. RESULTS: The ultrastructure of the explant of the ciliary nerve co-cultured with HSV showed that the viral capsid acquired a viral envelope and viral core, and a capsid and inclusion body within the nucleus. The enveloped virus was scattered within the vesicles of the cytoplasm. The virus-like particles were identified at the axonal fibers. CONCLUSIONS: The co-culture of the explant of the ciliary nerve and HSV showed the replicative process of the HSV within the cultured cell. The virus-like particles within the axon showed the evidence axonal transport of the virus under culture conditions.
Subject(s)
Axonal Transport , Axons , Capsid , Cells, Cultured , Coculture Techniques , Cytoplasm , Herpes Simplex , Herpesvirus 1, Human , Inclusion Bodies , Microscopy, Electron, Transmission , Nerve Growth Factor , SimplexvirusABSTRACT
Crystal HRP was applied to the cut ends of the left infraorbital nerve and branches of the facial nerve of 34 adult mice. Then the animals were killed and perfused, and HRP solution was applied to the cut ends of the root of the right facial nerve and the right maxillary nerve. Frozen sections of the brain stem and the trigerminal ganglia were made and reacted with BDHC, TMB or DAB method for light and electrom microscopic examination.Under light microscopy, the neurons labeled when alive in the left trigerminal ganglion and the nucleus of the left facial nerve showed that more cell bodise but less nerve fibers were labeled. The reaction products with all the 3 methods in thess cells were granular. The neurons labeled after death in the right trigerminal ganglion and the nucleus of the right facial nerve showed that more fibers but less cell bodies were labeled. The reaction products in these cells were also granular with BDHC and TMB methods but diffuse with DAB method.Under electron microscopy, there were electron-dense positive products in all the cells labeled either when alive or after death. In those cells labeled when alive, the products were found in the round or oval secondary lysosomes or multive-sicular bodies, while in those labeled after death, they were mostly found on one side of vacuoles without membrane boundary. In the fibers labeled after death, many large or small positive granules were found in the axons and many minute positive granules in the myelin lamellae especially on their inner surface in contact with the axons.
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The purpose of this research work is to find out the longitudinal distribution of the preganglionic neurons which project to the superior cervical sympathetic ganglion (SCSG). Experiments were performed on 12 adult rabbits and a monkey. Under sodium pentobarbital anesthesia, 20 microliters of 10% HRP were injected slowly into the rabbit's SCSG. In the monkey, 20 microliters of 15% HRP was injected.After a postoperative survival time of 3～6 days, the animals were perfused through,the ascending aorta with a cold fixative mixture composed of 2% paraformaldehyde and 1.25% glutaraldyhyde in 0.1mol phosphatebuffer at pH 7.4. The spinal cord segments C_1～L_3 were cut serially in transverse plane on a cryostat at 48 micra.The HRP reaction product was demonstrated according to Mesulam's (1976) benzidine blue reaction method, and counterstained with neutral red.HRP labeled neurons in the spinal cord were located exclusively on the side ipsilateral to the injected SCSG. The total number of labeled cells were 7908 in 12 rabbits, but the number of labeled cells varied from animal to animal. The highest amount was 1690 (423~#) and the lowest amount was 71 (425~#). The longitudinal distribution of the labeled cells in 12 rabbits was 12 segaments of the spinal cord (C_6～T_9), but the largest proportion (86.18%) of them was concentrated from T_1 to T_4, especially at the level of T_2 and T_3 (56.22%), and with a peak at T_2 (29.10%).In cross section of the spinal cord. HRP-labeled cells were concentrated in four cell groups, they are: nucleus intermediolateralis pars principalis (ILp), nucleus intermediolateralis pars funicularis (ILf), nucleus intercalatus (IC) and nucleus intercalatus pars paraependymalis (ICpe). The latter is subdivided into dorsal portion and ventral portion. HRP positive cells were mainly located in the ILp, In 12 rabbits about 92.99% cells were located in it. A small portion of labeled cells(6.25%) were seen in the ILf. A few labeled neurons could be detected within,the IC (0.68%) and ICpe (0.08%). Furthermore, occasionally, very few labeled cells were found at the dorsol portion of the anterior horn.In the monkey, generally speaking, the pattern of the distribution of labeled cells was the same as the rabbit.