| Dorsal root ganglion(DRG)cells are the primary afferent neurons of algaesthesis of trunk and extremities.DRG connects the spinal cord with internal and external environment of the organism.It not only transmits and accommodates sensations but also accepts and conveys nociception of the organism,so it plays an important role in the origination and maintenance of neuropathologic pain.Adriamycin,a broad-spectrum anthracycline antibiotic and anticancer therapeutic agent,has obvious neurotoxic effect and autofluorescence.In the recent years,it was demonstrated that intravenous injection adrinmycin can selective destroy the PNS sensory ganglion,it also cause the DRG cells necrosis but not apoptosis.Also it has highly affinity to the dorsal root ganglion and result in the degeneration of the DRG cells.So it was used in the clinical to treat the neuropathic pain which was named selective chemo-ganglionectomy.In the post experiment,we demenstrated that intervetebral foramina injiection with high concentration of adriamycin can lead the dorsal root ganglion cells to death,it also can result the sensory and motor function changes of the dermatome corresponding to the dorsal root ganglion.ObjectiveBuild the spared nerve injury rat model of neuropathic pain.Then observe the effection of the dorsal root ganglion by using doubel-label immunofluorescence after intervetbral foramina injiection with 0.25%,0.5%,0.75%,1.0%concentration of adriamycin。And test the algesthesia behavior changes of the rats before and after administration.The final purpose was to definite the death property of the SNI rats' dorsal root ganglion cells and to provide evidence for using the chemo-ganglionectomy to treat the neuropathic pain in the clinical.MethodAdult male Sprague-Dawley rats weighing 200g on the day of surgery were used in this study.The method of inducing nerve injury using the Decosterd method.After building the spared nerve injury model,all of the SNI rats were randomly assigned to five groups,respectively named control group(n=20),0.25%ADM group(n=20), 0.5%ADM group(n=20),0.75%ADM group(n=20),1.0%ADM group(n=20).After celiac anaesthesia with 10%chloraldurat(0.3ml/100g),the margin of left L4,L5,L6 intervertebral foramina of the rats were exposed under the condition of sterility. Then intervetbral foramina injection with 5μl adriamycin at the concentration of 0.25%,0.5%,0.75%,1.0%into the foamina of the rats correspongding to the groups.The control group rats were injected with NS into the foamina.The rats were then tested the day after surgery or administration and once a week for 4 weeks including the latency of withdrawal to the radiant heat stimulus,acetone test and pin-prick test.Then randomly dislodge L4,L5 DRG of the left side of 3 rats respectively from the five groups at the day 3,7,14,21,28 after administration.The L4 DRG was stained with HE to observe morphologic change of DRG cells using the light microscope.Then used the double-label immunofluorescence to observe the DRG cells' fluorescent light by laser scanning confocal fluorescence microscope.Results1.The pain behavior before administrationAll the spared nerve injury rats developed qualitative signs indicative of a marked sensory hypersensitivity of the hindpaw ipsilateral to the nerve injury.This was clearly present 24h after the surgery,appeared to reach a peak at about 2 weeks,and continued for as long as the animals were monitored.The animals refrained from weigh bearing on the affected paw and modified their stance accordingly,with eversion of the root.If contact was inadvertently made,either at rest or during locomotion,a prolonged flexion of the hindpaw was generated.Development of a sudden sustained spontaneous withdrawal in the absence of any stimulus of the hindpaw was present intermittently in most animals,the paw being held in a flexed position with paroxysms of smaller flexion.No autotomy or body weight loss was observed,nevertheless during the study and the animals' grooming.2.The pain behavior after administrationCompared to the control group,the other four groups' rat injected with different concentration adriamycin exhibited reversed pain behavior,including increased paw-withdrawal latency to the radiant heat stimulus,decreased paw withdrawal duration to acetone and pin-prick stimulus.The longer time observation of the 0.25%ADM and the 0.5%ADM group rats suggested that 0.25%ADM group rats' pain behavior rebound to pre surgery level at third week.And it was not advanced at the fourth week.However,the 0.5%ADM group rats rebound to pre surgery level were more early than 0.25%ADM group rats.The former developed hypoalgesia, including increased paw-withdrawal latency to the radiant heat stimulus,no response to the acetone and pin prick stimulus.High concentration groups(0.75%and 1.0%) rats rebound to pre surgery level at day 3 after administration.The longer time observation results suggested that high concentration group rats exhibited hypoalgesia at second week after administration.In our study,we did not observed obviously motor dysfunction and extremity palsy in the 1.0%ADM group rats.3.Histological pathological change of DRG cells(1)The results of paraffin section with HE stainAs for the group with 0.25%adriamycin,light microscopical study of DRG sections revealed hydropic degeneration,and the loss of Nissl's body at the second week.Karyopyknosis,cytoplasmic clarifixation and cellular necrosis were observed at the fourth week.However cellular universal degeneration,crimpy cytoplasmic, fuzziness and loss of Nissl's body were seen 2 weeks after injection with 0.5% adriamycin,moreover karyopyknosis and cellular necrosis in a small number of DRG were simultaneously seen.With that,karyopyknosis,karyolysis,satellite phenomenon and swallowing ganglia phenomenon were seen at the fourth week. Despite of cellular universal degeneration,karyopyknosis karyorrhexis and cellular necrosis were seen in a portion of DRG cells 2weeks after injection 0.75%and 1% adriamycin group.At the fourth week,mass cellular necrosis and replacement of hyperplastic neurogliocyte and connective tissue were seen.There was several cells degeneration in the dorsal root ganglion on the rats of the control group.(2)The results of double-label immunofluorescenceBy using the laser scanning confocal fluorescence microscope,we observed the positive cells expressed Bcl-2 or Bax.Compared to the control group,the positive cells expressed Bcl-2 or Bax decreased as the ADM concentration increased.Even in the same group,the positive cells expressed Bcl-2 or Bax decreased as the time longer. However,the control group DRG cells expressed Bax were so rare.ConclusionIntervetebral foramina injection with different concentration adriamycin into the dorsal root ganglion corresponding to the nerve injury can reverse the pain behavior of the SNI rats.It also can result in the DRG degeneration.The results of double-label immunofluorescence suggested that Bcl-2 and Bax were expressed in the dorsal root ganglion cells during the procedure of the cell degeneration.And the positive cells expressed Bcl-2 or Bax decreased as the ADM concentration and time increased.It suggested that apoptosis may contribute to the DRG cells' death.Or it may be describe that the lower concentration ADM can result the DRG cells apoptosis,the higher concentration ADM can result the DRG cells necrosis.
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