Changes Of Brain Pain Pathways In Rat Models Of Neuropathic Pain And Opioid-Induced Hyperalgesia:A FMRI Study

Objective To explore the functional changes of brain pain pathways in two relevant abnormal pain states: neuropathic pain and opioid-induced hyperalgesia;to investigate whether there are some underlying associations between the two pathological conditions in fMRI.Method 1.Groups and models of experiment:(1).Twenty Male SD rats were randomly divided into 2 groups(n=10):Each animal was subjected to a SNI operation(SNI group,n=10)or to a sham operation(SHAM group,n=10)involving the same extent of skin incision and muscle dissection.After the animals were anesthetized with isoflurane(5%induction and 1.5% maintain)and O2,the tibial and common peroneal branches of the sciatic nerve were exposed,and then the ligation(with 5-0 sterile suture silk)and transaction were conducted,leaving the sural nerve intact.In the sham control operation,the animals' nerves were exposed until visible as in the SNI procedure,but they received no further manipulations.Wound closure was performed with 3–0 sterile suture silk followed by cutaneous application of antibiotic ointment.(2).Twenty Male SD rats were randomly divided into 2 groups(n=10): OIH group(opioid-induced hyperalgesia)or CON group(control).Morphine injections were applied in OIH group rats(10mg,bid,9am?6pm)and the same dose of saline were administrated to CON groups,lasting for eight days until the day before fMRI scanning.2.Behavior test:(1).von Frey test: All von Frey testing was conducted in a lit and quiet room between the hours of 08:30 and 18:00.In the SHAM and SNI groups,von Frey test were performed at 2 days prior to surgery(pre-injury)and 3,5,7,9,11,13 days post-injury until the first fMRI scanning.Then,the test was performed every two weeks.One day before and three days after the first morphine injection,von Frey tests were administrated in OIH group and CON group rats to detect the 50%MWT.(2).Every morning 30 mins before and 1h after morphine injection,the tail flick test was performed in OIH group rats using a 52? water bath.3.The first fMRI scan: At the time point of two weeks after the operation when the OIH group had administrated morphine for eight days,the four groups were subjected to fMRI scanning,the main process of which was task-state scan.Being anesthetized by isoflurane(5%induction and 1.5%maintain),the rats were fixed on the scanning platform with a water bath to maintain the body temperature,and their heart rates,breath and rectal temperature were continuously being monitored.In the task-state scanning,a nociceptive mechanical stimulation was applied to the lateral left plantar of rats by long-range control.4.The second fMRI scan: After the first fMRI scanning,the rats of OIH group and CON group were sacrificed under anesthetic state,while the rats of SHAM and SIN groups were continued to feed.The long-term fMRI scanning(the second fMRI scanning)were processed at two months after operation with the same procedure as the first time.Results 1.(1).For the von Frey test,ANOVA analysis yielded a main effect of Group(F=60.511,P<0.001)and indicted the Side(F=21.137,P<0.001)and time(F=4.046,P=0.001)also made significant effects,with SNI rats showing a decreased 50%MWT for the ipsilateral hindpaw compared to SHAMs.In addition,a significant Group×Side interaction were proved.Two-tailed tests reveals there were no evident differences between the two groups at the pre-injury time point(P>0.05);however,at the post-injury time points of 3,5,7,9,11,13 days after operation,SNI rats showed significantly lower 50%MWT(P<0.001)for the ipsilateral hindpaw compared to SHAMs.For contralateral hindpaw,there were no significant differences between the SNI and SHAM groups at all time points except the fifth day after operation.(2).At the time point before morphine injection,t-test showed no significant group difference in 50%MWT between the CON and OIH,while,OIH rats demonstrated significantly lower 50%MWT for lateral hindpaw compared to CONs since the time point of the four day after morphine application,showing the opioid-induced hyperalgesia established.(Fig 2)2.The %MPE in the OIH group was the highest in the subcutaneous injection of morphine at the first day,which indicted the analgesic effect of morphine was the strongest.At the third day,the %MPE of OIH group had an evident decrease(P <0.001),and the analgesic effect of morphine gradually decreased in the remaining days of morphine administration,concluding that the morphine efficiency significantly decrease.(Fig 3)3.Results of the task-state scans:(1).OIH and CON group(Fig 4A): In the imaging of CON group,relative to the baseline,there are negative activities of nucleus accumbens,caudate putamen,septal nucleus,bed nucleus stria terminalis.There are mostly positive activities in OIH rats in response to the stimulus,including the bilateral cingulate cortex,bilateral primary and second motor cortex(M1?M2),the bilateral primary somatosensory cortex(S1),bilateral hippocampus,some right thalamic nuclei(the ventral,dorsal and posterior nuclei),bilateral retrosplenial cortex,paraventricular nucleus,habenular nucleus and pineal gland.(2).SNI and SHAM groups 2 weeks post-operation(Fig 4B): In response to a nociceptive mechanical stimulus,in SHAM group,there are negative activities in nucleus accumbens,caudate putamen,septal nucleus,bed nucleus stria terminalis.In SNI group,there are mainly positive active brain regions including the bilateral cingulate cortex,left(ipsilateral to injured hindpaws)M1?M2,the left(ipsilateral)S1,the left insular cortex,bilateral hippocampus,bilateral dorsal thalamic nucleus,bilateral retrosplenial cortex,and pineal gland.(3).SNI and SHAM groups 2 months post-operation(Fig 4C): There were no evident activities of brain in response to nociceptive stimulus in SHAM rats at the 2 months post-operation.In the SNI group,relative to baseline,the nociceptive mechanical stimulus application to the left hindpaw evoked positive activities in retrosplenial cortex,right(contralateral)secondary visual and auditory cortex.Conclusion 1.Brain functional changes stemed from SNI operation was a sequential process,in which the fMRI data at short-term time point(post-injury two weeks)showed evident differences to it at long-term time point(post-injury two months).At the short-term time point,there were many brain regions hyperactive involved in classic pain matrix(PM),including cingulate cortex,primary and second motor cortex,primary somatosensory cortex,insular,hippocampus,thalamus,while,at the long-term time point,no significant PM-relevant regions activities were found.According to our data and previous researches,the short-term PM hyperactivity may gradually change to a dysfunctional and disinhibition state.In other words,in the latter state,the normal PM interaction may be disturbed and some brain regions important to modulate nociceptive signals may be inhibited,resulting in the hyperalgesia and allodynia.Notably,the hyperactive cortices at 2 weeks were at ipsilateral side of the stimulus,indicating the normal pain pathways had transferred to the contrary side.2.After eight days morphine injection,the established OIH were associated with some PM's hyperactivities in fMRI,which had some similarities to the imaging of SNI at the short-term.Both had cingulate cortex,S1,M1?M2,thalamic nuclei,hippocampus hyperactivities,which were main components of PM.The most obvious differences between them were:(1).The hyperactive PM of OIH group was mainly at the contralateral side to stimulus with less activities at the opposite side,while the SNI group was exclusively at the ipsilateral side,indicating the SNIs had abnormal pain pathways transfer.(2).There were some pain-relevant thalamic nuclei hyperactivities in OIHs,such as ventral and posterior nuclei,which couldn't find in SNIs.(3).There was ipsilateral insular cortex hyperactivity in SNIs but not in OIHs.Based on the previous literatures,the similarities of them might represent an inflammation state of the central nervous system.The SNIs with abnormal ipsilateral activities might be because the SNI produced interruptions to normal pain pathways.In general,the SNI can contribute to early abnormal pain pathway transfer and lead to a sequential change of PM in which the PM hyperactivities in the short-term turn to a possible dysfunction and disinhibition.In addition,the OIH brings out PM hyperactivities which have some resemblances with SNIs in short-term but more consistent with the physical pain pathways.