Effects Of Enriched Environment On Pain After Thalamic Hemorrhage And Its Mechanism

BackgroundEnriched environment(EE)was first proposed by Canadian scientist Hebb in1947.He found that EE rats had significantly higher learning and memory abilities.EE is an emerging non-invasive method of low-cost rehabilitation nursing,which is different from traditional drug or surgical treatment.It is based on benign changes in a variety of cognitive,sensory,social interactions and movements,and has been shown to have favorable therapeutic effects for many diseases.In animal model studies,EE has been extensively studied in central nervous development.Central nervous system injuries or diseases(stroke,spinal cord injury,multiple sclerosis,etc.)often cause central pain,which is usually characterized by a loss of motor function.However,in the model of central nervous system disease,studies on EE have focused more on motor recovery and the potential mechanism of nerve regeneration,with little attention paid to post-stroke pain,and its complications such as anxiety,depression and decreased learning and memory ability have also been often ignored.Stroke is one of the major diseases threatening human health,especially intracerebral hemorrhage(ICH)with high disability rate and mortality.70% of stroke patients will have chronic pain within six months,and central post-stroke pain(CPSP)accounts for about 10% of the chronic pain,which is commonly seen in thalamic lesions.Thalamic pain is a typical CPSP.The ventral thalamus nucleus group carries signals from the peripheral nervous system to specific parts of the cerebral cortex.In particular,the ventroposterior lateral nucleus(VPL)receives signals mainly from somatosensory nuclei and transmits them along the axons of dorsal column nuclei(DCN)across the midline to the medial thalamus of the opposite side,terminating in the VPL.VPL neurons project thalamic-cortical axons to the somatosensory cortex of the posterior central gyrus(SⅠ area),and neurons in the SⅠ cortex project to the SⅡ area.There are few studies on the pathological changes of ultrastructure after thalamic hemorrhage,including changes in neurons,myelin sheath,synapses and microglia.The observation of ultrastructure deepens the understanding of the disease,including the basic lesions,etiology and pathogenesis.CPSP is a central neuropathic pain syndrome that occurs after stroke.It is directly caused by cerebrovascular diseases that damage the somatosensory system of the central nervous system.It is mainly manifested as pain and paresthesia in the body parts corresponding to the brain injury area.The pain lasts for a long time,and it is intense,whose nature is difficult to describe.It can be induced by mechanical or temperature stimulation(especially cold stimulation).The prevalence of CPSP in stroke patients ranges from1% to 12%.Patients cannot effectively cooperate with functional rehabilitation training because it is unbearable,which seriously affects the quality of life of patients,and can produce depression,anxiety and other abnormal emotions that seriously affect mental health,accompanied by cognitive,language and emotional disorders,but there is no effective treatment at present.EE has a certain positive effect on the treatment of pain.Studies have shown that EE can improve the pain threshold of neuropathic animal models by reducing neuroinflammation,reducing neuronal excitability,and regulating the release of neurotrophic factors and chemokines.However,no studies on EE in CPSP have been reported.CPSP appeared in the process of secondary injury of intracerebral hemorrhage(ICH).During the dissolution and decomposition of hematoma,harmful metabolites(including thrombin,iron overload and heme,etc.)are continuously produced,which produce strong cytotoxicity to the surrounding cells of hematoma,causing inflammatory reaction and oxidative stress,etc.,and play a key role in the generation and development of CPSP.Studies have shown that neuroinflammation is an important part of ICH secondary injury.It mobilizes the immune system,and activates relevant cell signaling pathways to promote the production of inflammatory cytokines,chemokines and other neurotoxic substances.Thus,it stimulates a series of inflammatory cascades,mediates the secondary damage of cells,and aggravates neuropathic pain.Microglia are widely distributed in the brain and are the main intrinsic dynamic immune cells,which have a dual role in pathological injury.Microglia have both pro-inflammatory and neuroprotective functions,depending on the changes of their activation state.It has been found that in CPSP animal models,microglia cells are in a state of aggregation and activation around the hemorrhagic foci.They can release a large number of cytokines and inflammatory mediators,thus causing hyperalgesia.Preliminary research of the research group and literature show that toll-like receptor 4(TLR4)is a classic innate immune receptor,which is mainly expressed on microglia in the central region.After the activation of TLR4,it recruits myeloid differentiation factor 88(My D88),which,after transduction,further causes the activation of nuclear transcription factor-kappa B(NF-κB),and then enters the nucleus to express inflammatory genes.These pro-inflammatory factors damage neurons,causing them to excite and amplify pain signals,which in turn cause the body to become allergic to painful stimuli.In conclusion,neuroinflammation plays a promoting role in the occurrence and development of CPSP,and TLR4 activation is the key point of microglia activation.Therefore,CPSP can be alleviated by reducing the activation of TLR4 receptor.However,it is not clear whether EE affects the activation of microglia to relieve pain after thalamic hemorrhage.In this study,through the application of EE in the treatment of pain after thalamic hemorrhage,we detected whether pain was relieved,and whether microglia activation and neuroinflammatory pathway proteins were decreased,analyzed the correlation between pain and microglia activation,and observed whether learning and memory ability and anxiety and depression-like mood were improved.Currently,there is a lack of clinical studies on EE in the treatment of pain.It is expected that EE can provide relevant theoretical basis and new directions for the treatment of CPSP,and it is very important to introduce the concept of EE into the rehabilitation treatment of pain patients and carry out clinical studies.Purpose1.Establish CPSP model of thalamic hemorrhage.Observe histological changes and ultrastructural changes around the hematoma.2.Elucidate the alleviating effect of enriched environment on pain after thalamic hemorrhage and its mechanism.Methods(1)A model of thalamic hemorrhage was established to observe the changes of histomorphology and ultrastructure around the hematoma.Mice were randomly divided into Sham group and CPSP group,and Evans blue was used for localization training according to the mouse brain atlas.Collagenase type Ⅶ-S was stereotaxically injected into the VPL region,and thalamus hemorrhage model was established.Changes in body weight,neurological function,mechanical pain and cold pain were measured at each time point.Fresh sections were performed on postoperative D1.The hematoma location and hematoma volume were measured.CV/LFB staining was used to detect demyelination changes at D3 after thalamic hemorrhage,and the degeneration of neurons around the hematoma was detected by FJC staining.Meanwhile,the ultrastructural changes of tissues adjacent to the hematoma were observed by transmission electron microscopy.(2)CPSP was treated with EE to evaluate the therapeutic effect of EE and explore its mechanism.Mice were randomly assigned to Sham group,CPSP group and treatment group(CPSP+EE group)after preoperative behavior elimination.Changes in body weight and neurological function,mechanical pain and cold pain were measured at each time point during the course of EE treatment.At D14 after treatment,changes in learning memory and anxiety and depression-like mood were detected.Changes in myelin sheath around hematoma were observed by CV/LFB,and changes in activation of microglia(Iba1)and astrocytes(GFAP)around injury were detected by immunofluorescence staining.The correlation between the pain production and the pain relief after EE treatment of D14 and the activated area of glial cells was analyzed.The protein expression of TLR4/My D88/NF-κB signaling pathway were detected by western blot(WB).Results(1)Two hours after microscopic injection of Evans Blue into VPL through a glass electrode,fresh sections were made and compared with the position and shape of the mouse brain atlas,and the position was accurate.After collagenase injection,one day after surgery,hematoma volume was calculated and compared with Sham group(P<0.05).Compared with Sham,CPSP group showed no significant changes in body weight and neurological function scores(P>0.05).Postoperative D1 mechanical pain began to be significantly increased(P<0.05),and cold pain was significantly increased(P<0.05)in postoperative D3,which lasted until D14.Compared with the Sham group,CV/LFB showed reduced neuronal myelination and demyelination around the hemorrhage site 3 days after surgery.Degenerated neurons were found around the bleeding foci in FJC staining compared with the Sham group.Immunofluorescence staining showed activation of microglia(Iba1)around the bleeding foci compared with the Sham group.The ultrastructural changes of brain tissue at D3 after injury were observed by transmission electron microscopy.Compared with the Sham group,red blood cells appeared around the hematoma,and the morphology of neurons and microglia were changed.In neurons and microglial cells,mitochondrial swelling,vacuolation and blurred ridge fracture,endoplasmic reticulum and Golgi swelling and fragmentation of organelle lesions were observed.The statistical results showed that compared with the Sham group,the number of myelin sheath around the hematoma was reduced(P<0.05),the myelin lamina was dilated and fluffy with the significant demyelination(P<0.05),and the number of synapses was reduced(P<0.05).Observe the phagocytic function of microglia cells after thalamic hemorrhage,the morphological changes of microglia cells and the pathological changes of organelles during this process.(2)The therapeutic effect of EE was tested,and the changes in the sensitivity to pain in the Sham group,CPSP group and CPSP+EE group were observed.EE improved the pain after thalamic hemorrhage,as well as the learning and memory disorders and anxiety and depression-like emotions after CPSP.Compared with the Sham group,mechanical pain in the CPSP group was significantly increased on postoperative D1(P<0.05),and cold pain was significantly increased on postoperative D3(P<0.05),which lasted until D14.In the CPSP+EE group,mechanical pain and cold pain were significantly improved on postoperative D7(the second day after EE intervention),which lasted until D14(P<0.05).The learning and memory ability was measured by Y maze and new object recognition test.Compared with Sham group,CPSP group showed learning and memory impairment on postoperative D14(P<0.05),which could be significantly improved after EE treatment(P<0.05).Elevated plus maze and open field tests were used to detect anxiety-like mood changes.Compared with Sham group,anxiety-like mood appeared on the 14 th day after surgery in CPSP group(P<0.05),and anxiety-like mood was improved in CPSP+EE group(P<0.05).Tail suspension and forced swimming tests were used to detect depression-like mood changes,and EE treatment improved depression-like mood on the 14 th day after surgery in the CPSP group(P<0.05).(3)EE improved demyelination after CPSP on the 14 th day after surgery,weakened glial cell activation,and reduced TLR4 signaling pathway protein expression.Compared with the Sham group,decreased myelination occurred 14 days after CPSP,and demyelination was improved after EE treatment(P<0.05).Compared with the Sham group,microglia and astrocytes were significantly activated in the CPSP group 14 days after surgery(P<0.05),and the activation of microglia and astrocytes decreased after EE treatment(P<0.05).Pearson product moment correlation/Spearmen rank correlation analysis showed that the production of pain was correlated with glial cell activation(P<0.05).The relief of pain after EE treatment was correlated with decreased activation of glia cells(P<0.05),except for 0.07 g mechanical pain(P=0.092),and the correlation between pain and microglia was stronger than that of astrocytes.WB test results showed that the protein expression of TLR4,My D88 and NF-κB were increased after CPSP compared with Sham group(P<0.05),and the protein expression was decreased after EE treatment(P<0.05).ConclusionsEE treatment alleviates pain after thalamic hemorrhage,improves learning and memory function and anxiety and depression-like behavior,and the mechanism may be inhibition of microglia activation,and reduction of TLR4 signaling pathway expression.