The Experimental Study About Thyroid Hormone Promoting The Remyelination In Remission Period Of Multiple Sclerosis

Multiple sclerosis (MS), the classical demyelination disease, is characterized by pathological changes of inflammation, demyelination and axon losing. The symptoms of multiple sclerosis include locomotor, sensory and cognitive dysfunction such as walking difficulty, fatigue, pain and memory decline. These syndromes are different in terms of the number and size of lesions and pathologies including:inflammation; demyelination and axonal injury; degree of remyelination and repair.In the nature history of prototypic relapsing-remitting MS, the remission period of symptoms is considered as the key period of remyelination and injured axons compensation. The demyelination axons would be rewrapped by remyelination, and the axons dysfunction was compensatory by rewrapped. So the remission period was considered as the remyelination period too. But the patients of the primary and the secondary progressive MS(PPMS and SPMS), the two special clinical types of MS, even do not have the remission period after the first or the second clinical attack, and they rapidly lead to severe disability and dysfunction.So the most conspicuous difference between the PPMS/SPMS and the classical relapsing-remitting MS was the remission period when remyelination takes place, and there may be something changed to prevent from the myelin regeneration and axon protection in PPMS and SPMS.In the other side, adult brains have the ability to generate oligodendrocytes, and the brains have the capacity to restore the damaged axons. However, remyelination is incomplete and eventually fails, so although the relapsing-remitting MS have the remission periods, the course of disease and pathology will go progressively and cumulatively. With the current anti-inflammatory and immunomodulatory treatments, the development of remyelination, neuroprotection and clinical disability have only minimal effects on multiple sclerosis.So the remission period is the key period regarding the myelin regeneration and axons protection. If we could find a way to keep adequate and functional remyelination and induce effective neuroprotection in this remyelination process period, the MS would be easy to recover and would improve the prognosis. Although the attack will happen next time, they will recover completely in the next remission period. This is also the new and widely-accepted remyelination and neuroprotection treatment strategies for the MS.In clinical course, there are some evidences indicating that the thyroid hormone may involved in the differences between the PPMS/SPMS and the classical relapsing-remitting MS. The recent study has found an abnormal thyroid hormone existing in the brain CSF of the MS patients. The MS patients have significantly higher TT4levels and higher TT4/rT3ratio in the cerebrospinal fluid (CSF). And the abnormal TH levels also have something to do with the clinical types of disease. However whether the abnormal TH level is the reason or the result, we don’t know yet. The above clinical course indicated that TH might play a very important role in the course of different clinical types of MS. Particularly, TH might affect remyelination process in the remission period.It is well established that TH is required for the normal timing of OPC differentiation and maturation, cell-cycle stopping mechanism, terminal differentiation and myelin production, stem cells differentiation and even transdifferentiation. Brain function and individual development are affected by thyroid hormone at an early age. And TH play a crucial role in the brain development, as hypothyroidism in childhood can lead to the cretinism. It is proved myelination is delayed in hypothyroid animals and accelerated in hyperthyroid animals too. To the myelination and myelin forming cell-oligodendrocyte, there are abundant evidences to show TH plays an important role in timing oligodendrocyte development and differentiation. Particularly, TH can implicate in oligodendrocyte differentiation and maturation via nuclear hormone receptors, and TH can act in the generation of positive signals for demyelination recovery.But, during the key period of the remission, how does TH affect the remyelination process? And is it necessary for the TH in the remyelination process? There are not many details especially on the TH deficit effects during the remyelination process.We use the cuprizone model, which had been used as models to study demyelination-associated diseases as well as remyelination processes. To imitate the remission period of MS, The CPZ model mice were given the diet containing0.2%cuprizone (bis-cyclohexanone -oxaldihydrazone, sigma) and available ad libitum for6weeks to induce critical demyelination, Then we withdrew the Cuprizone diet and turned to normal diet for2weeks to induce remyelination. The remyelination for2weeks was imitated the remission period of MS.In this period we manipulated thyroid hormone with the PTU (6-propyl-2-thiouracil) and T3agents, used0.15%6-propyl-2-thiouracil (PTU) in drinking water to inhibit mouse’s TH secret, and injected intraperitoneally exogenous T3hormone (300ng/g, in PBS, body weight) to increase mouse’s TH level. In this present study, we used immunohistochemistry, behavioral tests, electron microscopy, western blotting to investigate TH effects on the behavioral performance; corpus callosum (CC) remyelination and axon protection.The results and findings can be divided into three parts.Part1:Thyroid hormone manipulation impacts the body weight and behavioral appearances during remyelination period.1. The animal groups and TH detected The CPZ mice were divided into4groups: CPZ+N, CPZ+PTU, CPZ+PBS and CPZ+T3groups. During the2weeks of remyelination, the CPZ+PTU group was given the PTU to inhibit the TH secretion, with the CPZ+N group as the control; the CPZ+T3group was given the T3IP to promote the TH, with the CPZ+PBS group as the control. The normal mice were also divided into4groups:N, N+PTU, N+PBS and N+T3groups. In the last2weeks, they were given the same interventions as the CPZ mice’s control.6weeks later, we checked the demeylination of the CPZ model mice by LFB and MBP staining. Significant demyelination was found in the CC of CPZ model mice, indicating that we have succeeded to build up the demyelination model.8weeks later, we detected the serum thyroid hormone level changes by radioimmunoassay. PTU in drinking water significantly inhibited the total serum T3and T4levels, and T3IP significantly enhanced the total serum T3level but accordingly inhibited the total serum T4levels by negative feedback. We succeeded to manipulate the thyroid hormone resulting in the serum TH levels changes during the remyelination period.2. Body weight monitoring To monitor the overall situation, we checked the mice body weight daily. The CPZ+PTU group had significant delay on the body weight recovery—the body weight only recovered to0.38±1.70g (CPZ+N:3.59±1.14g; P<0.05) in the end. Interestingly, the CPZ+T3group did not enhance the body weight recovery compared to the CPZ+PBS group—the body weight only reached2.14±1.09g, but still recovered better than the CPZ+PTU group (P<0.05). The TH played a very important role in the overall situation recovery during the remyelination period.3. Motor coordination and balance ability monitoring To monitor the motor coordination and balance ability of mouse, we recorded the maintaining times of mice on the accelerating and high speed rod stick. When withdrew the CPZ diet and turned to normal diet to induce the remeylination, there was notable increase in the maintaining times in both CPZ+N and CPZ+PBS groups. However, the recovery of the CPZ+PTU group was restrained and the maintaining times only reached84.88±10.96s(CPZ+N:105.67±20.72s; P<0.05). Cheerfully, the maintaining times of the CPZ+T3group was enhanced, which got to120.10±14.70s(CPZ+PBS:106.13±14.38s; P<0.05). During the remmyelination period, TH deficiency affected the recovery of motor coordination and balance ability, while increasing TH level promoted the motor function and balance ability recovery.4. Spontaneous activity and exploratory behavior detecting To evaluate the spontaneous activity and behavioral response to the new environment, the open-field test was carried out to assess the action of the mice. The spontaneous activity and exploratory behavior of the CPZ+PTU group was significantly reduced—the travel distance of the CPZ+PTU group was only15803.98±1241.49mm(CPZ+N:20396.96±2047.39mm; P<0.05). However, the response of the CPZ+T3group was slightly enhanced, with the traveled distance increasing to21167.86±1646.75mm(CPZ+PBS:19747.35±1682.11mm; P>0.05). There was no difference between the four groups in terms of the centre-area distances and centre-area time ratios, which kept about3.7%and2.4%respectively. Those results indicated the spontaneous activity changes may mainly be caused by actional function changes, but not be affected by the exploratory behavior habit changes.Part2:Thyroid hormone manipulation impacts the remyelination and oligodendrocytes lineage cells during remyelination period.1. TH promoted the CC remyelination We tested the remyelination changes by LFB staining, MBP immunochemistry staining and western blotting. Compared with the CPZ+N group, the myelin staining of the CPZ+PTU group was loose and thin; and the myelin protein expression were significantly decreased (P<0.01). However, the myelin staining of the CPZ+T3group was restored to be thick and dense, and the myelin protein expression significantly increased (P<0.01). Those findings indicated that during the remyelination period, TH deficiency would inhibit the myelin recovery; however, improving the TH level could enhance the myelin recovery.2. TH promoted the increase of myelinted axons. To detect the ultrastructure changes, we made use of the electron microscopy to check the CC area. The CPZ+PTU group shew few myelinated axons and large nude axons existing (P<0.01). But the myelin thickness was no different than the CPZ+N group. However, the myelinated axons of the CPZ+T3group significantly increased (P<0.01). But the myelin thickness didn’t increase compared to the CPZ+PBS group. In the remyelination period, the TH manuipulation had no effect on the myelin thickness, but affected the quantity of the myelinated axons.3. TH promotes the oligodendrocyte linage cells proliferation.By immunohistofluorescence assay, TH in the remyelination period impacted all the oligodendrocyte phases in the CPZ remyelination model, including the mature, the pre-mature and the precursor phases. There was even no OPCs proliferation in the SVZ area due to the lack of TH. Compared to the CPZ+N group, there were only21.3%of OPC cells,76.7%of pre-mature and58.7%of mature cells presented in CC. However, TH played an incentive role to promote OPC proliferation and differentiation. The three phases of the oligodendrocyte all increased in the CC area, especially the SVZ had a remarkable proliferation of OPC. TH promoted remyelination may by promate the oligodendrocyte lineage cells proliferation and differentiation.Part3:Thyroid hormone manipulation impacts the axons condition and astrocytes but doesn’t affect the microglias in remyelination period.1. TH promotes the axons’being rewrapped and survival We detected the axons conditions by high magnification confocal immunofluorescence and electron microscopy assays. Compared to the CPZ+N group, the CPZ+PTU group showed large areas of NF200+staining and small areas of MBP+staining. The red NF200staining was continuous and linear, and it extended along the axon. This phenomenon indicated the nude axons after demyelination. The electron microscopy graph showed the axons of the CPZ+PTU group had a bad survival conditions with the enlarged mitochondria. However, The CPZ+T3group showed large areas of MBP staining and small areas of NF200staining, and the red NF200staining was no longer continuous and linear, which indicated the axons were rewrapped by remyelination. The electron microscopy graph showed the axons of the CPZ+T3group had an improved survival condition, with the mitochondria returning to the normal level. In the remyelination period, TH deficiency would delay the nude axons’being rewrapped and cause the bad survival condition, and improving TH level would promote the nude axons’being rewrapped and improve the survival condition.2. TH reduces the astrocytes proliferation and activation but dosen’t affect the microglias We detected the astrocytes changes in CC area by GFAP immunochemistry staining and western blotting. The CC area of CPZ+PTU group has got large astrocytes proliferation and the morphous of those astrocytes were large and dense with many dendrite. And the CPZ+PTU group showed large amount of GFAP protein too. However the CC area of the CPZ+T3group had few astrocytes proliferation and the morphous of those astrocytes were small and thin with few dendrite. And CPZ+T3group showed small amount GFAP protein. We also detected the microglia changes by F4/80immunochemistry staining. In the remyelination period, the microglia decreased in all groups—it had nothing to do with the TH changes. In the CPZ remyelination model, TH improvement could help the active astrocytes turn to normal morphous and reduce the proliferation. On the contrary, the TH deficiency caused much more active astrocytes in demyelination area. This maybe another factor to inhibit the myelin regeneration.In the clinical course of MS, not much attention was put on the remission period, as the remission period was the key period of remyelination takeing place. Promoting effective remyelination and neuroprotection would improve the MS prognosis. Our works indicated that the body’s thyroid hormone level had a critical influence on the remyelination. TH deficiency would delay the remyelination and motor funcation recovery. On the contrary, promoting the TH level would significantly increase the remyelination and motor funcation recovery. This research provided a new insight to TH’s affects on the remyelination process and emphasized the treatment should focus on the remission period for MS cure.