Establishment of longitudinal transcranial stimulation motor evoked potentials monitoring of the forelimbs and hindlimbs in an ischemic stroke rat model

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Establishment of longitudinal transcranial stimulation motor evoked potentials monitoring of the forelimbs and hindlimbs in an ischemic stroke rat model

https://doi.org/10.21203/rs.3.rs-1954637/v1

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Evaluation of motor function ischemic stroke rat models included qualitative assessments such as the modified neurological severity score (mNSS). However, mNSS cannot evaluate the function of forelimbs and hindlimbs separately. We quantitatively assessed motor function in a middle cerebral artery occlusion (MCAO) rat model of ischemic stroke. We recorded transcranial stimulation motor evoked potentials (tcMEPs) from MCAO rats and measured the changes in latency and amplitude at the forelimbs and hindlimbs up to 28 days after stroke. All MCAO subjects showed hemiparesis. Their mNSS scores were highest on day 1 and then decreased over time. The score change curve of the tcMEPs amplitude in the hindlimbs was inversely associated with mNSS, while tcMEPs amplitude in the forelimbs was lowest on day 14 and then increased with time course. The latency of tcMEPs in the forelimbs and hindlimbs remained almost unchanged during the follow-up period. Our results showed the differences in tcMEPs amplitude recovery times between the forelimbs and hindlimbs after MCAO, which emphasizes the importance of separately evaluating forelimbs and hindlimbs in post-ischemic stroke models. This minimally invasive and longitudinal quantitative method could be useful for further research on diseases and neurogenesis.

Ischemic stroke is a major global disease with high incidence and mortality rates ^1,2. Among symptoms of ischemic stroke at major vessels, motor dysfunction is a serious presentation mainly caused by the middle cerebral artery occlusion (MCAO). In animal models of ischemic stroke, MCAO is the most common method for investigating the neurological and histological outcomes and/or pathological mechanisms of the condition ^3,4. For the evaluation of motor function in ischemic stroke rat models, including the MCAO model, “qualitative” assessments by behavioral evaluation such as the modified neurological severity score (mNSS) ^5,6, cylinder test ^7–10, and the Rotarod test ^11,12 have been used. However, “quantitative” methods have been strongly desired for an accurate evaluation of ischemic stroke rat models. It has been reported that transcranial stimulation motor evoked potentials (tcMEPs) are highly reproducible in mouse models ¹³. Few reports have documented the utility of tcMEPs in rat models, some of which performed longitudinal evaluations in rat spinal cord injury models ^14–16. We previously found that tcMEPs were not reproducible for adult rats because of skull thickening during growth ¹⁷. Therefore, we developed the technique of longitudinal tcMEP evaluations in a rat spinal cord injury model via the skull thinning method, and finally established it as a highly reproducible tcMEP recording method for the hindlimbs ^17,18.

To date, there have been no reports of long-term recordings of tcMEPs in a rat model of ischemic stroke. Moreover, there have been no reports of long-term recordings of tcMEPs for the forelimb. The assessment of forelimb function might be unnecessary for spinal cord injury models, whereas it is essential for ischemic stroke models with forelimb and hindlimb hemiparesis. We applied our skull thinning method ¹⁷ in an MCAO rat model to determine the longitudinal quantitative evaluation of motor function by tcMEPs in the fore and hindlimbs. Here, we established the highly reproducible tcMEPs method in the MCAO rat model.

Electrophysiological Recordings

Representative recordings of tcMEPs in the forelimbs of rats plotted before and at 1, 7, 14, 21, and 28 days after the MCAO procedure are shown in Figure 1a. The time course of tcMEPs amplitude in the forelimbs is shown in Figure 1b. Respectively, the mean (standard deviation) of tcMEPs amplitude and the 95% CI were 9661 (1448), 7995 to 11326 μV before the MCAO procedure, 6189 (3803), 1816 to 10562 μV at day 1, 5794 (3837), 1383 to 10205 μV at day 7, 5017 (2390), 2270 to 7764 μV at day 14, 8407 (4100), 3694 to 13121 μV at day 21, and 7073 (1525), 5320 to 8827 μV at day 28. Forelimb amplitude decreased on day 1 and did not recover until day 14 after which it increased over time, changing significantly between recording dates over the postoperative course (p < 0.05, one-way ANOVA). The forelimb amplitude was less than 50% of baseline between day 1 and day 14, and then recovered to 81.3% of baseline on day 28 after ischemic stroke.

The time course of tcMEPs latency in the forelimbs is shown in Figure 1c. Respectively, the mean (standard deviation) of tcMEPs latency and the 95% CI were 0.86 (0.24), 0.56 to 1.16 ms before the MCAO procedure, 0.82 (0.23), 0.54 to 1.10 ms on day 1, 0.74 (0.15) 0.55 to 0.93 ms on day 7, 0.84 (0.25), 0.53 to 1.15 ms on day 14, 0.66 (0.08), 0.55 to 0.76 ms on day 21, and 0.78 (0.48) 0.34 to 1.21 ms on day 28. The tcMEPs latency in the forelimbs did not change significantly during the follow-up period (p ≥ 0.05, one-way ANOVA).

Representative recordings of tcMEPs in the hindlimbs of rats before and at 1, 7, 14, 21, and 28 days after the MCAO procedure are shown in Figure 2a. The time course of tcMEPs amplitude in the hindlimbs is shown in Figure 2b. Respectively, the mean (standard deviation) of tcMEPs amplitude and the 95% CI were 10103 (3322), 7289 to 17357 μV before MCAO procedure, 2990 (1629), 919 to 5632 μV at day 1, 5529 (4840), 2779 to 16297 μV at day 7, 7797 (3489), 4687 to 14974 μV at day 14, 8367 (1919), 5087 to 11039 μV at day 21, and 8678 (1444), 7250 to 11263 μV at day 28. The amplitude in the hindlimbs was lowest on day 1 and then increased with time, changing significantly between recording dates after the MCAO procedure (p < 0.01, one-way ANOVA). The hindlimb amplitude was lowest on day 1 (29.6% of baseline) and recovered to 85.9% of baseline on day 28. The time course of tcMEPs latency in the hindlimbs are shown in Figure 2c. Respectively, the mean (standard deviation) of tcMEPs latency and the 95% CI was 5.16 (0.29), 4.80 to 5.52 ms before the MCAO procedure, 5.42 (0.30), 5.04 to 5.80 ms on day 1, 5.14 (0.77), 4.18 to 6.10 ms on day 7, 4.98 (0.33), 4.56 to 5.40 ms on day 14, 6.04 (1.56), 4.11 to 7.97 ms on day 21, and 5.22 (0.27), 4.89 to 5.55 ms on day 28. The tcMEP latency in the hindlimbs did not change significantly during the follow-up period (p ≥ 0.05, one-way ANOVA).

Neurological functional recovery in the MCAO rat model

We assessed neurological function after the MCAO procedure using mNSS. After the MCAO procedure, all rats had left incomplete hemiparesis. The time course of the mNSS score is shown in Figure 3. Respectively, the mean (standard deviation) of mNSS score and the 95% CI were 9.40 (0.89), 8.29 to 10.51 at day 1, 8.20 (0.45), 7.64 to 8.76 at day 7, 6.80 (0.45), 6.24 to 7.36 at day 14, 5.40 (0.55), 4.72 to 6.08 at day 21, and 4.80 (0.45), 4.24 to 5.36 at day 28. The mNSS score was highest on day 1 and improved over time, changing significantly between recording dates after MCAO procedure (p<0.01, one-way ANOVA). The score change curve of mNSS was inversely associated with that of tcMEPs for hindlimbs, but poorly associated with that of tcMEPs for forelimbs.

Histological analysis

The area of cerebral infarction after the MCAO procedure was evaluated histologically using hematoxylin and eosin staining. As shown in Figure 4, the cerebral infarcts were observed in the reflux area of the middle cerebral artery, which included the sensory-motor overlap area of the forelimb at the infarction core and the hindlimb at the infarction marginal area ¹⁹. In addition, we confirmed that the brain parenchyma was not damaged by skull drilling and electrical stimulation.

In this study, we established a method for recording tcMEPs longitudinally up to 28 days after a cerebrovascular incident in an ischemic stroke rat model in both hindlimbs and forelimbs. There has been no report of long-term recordings of tcMEPs in an MCAO rat model. One study which achieved short-term recordings ²⁰. In that report, tcMEPs recording were performed by using stainless steel screw needle electrodes continuously implanted in the skull ²⁰. However, this method was highly invasive and carried a high risk of infection, making a long-term recording of tcMEPs difficult. For the long-term recording of tcMEPs in rat models, it is necessary to reduce the level of invasiveness. Our method of using percutaneous needle electrodes for stimulation and recording is a minimally invasive and simple procedure that allows repeated electrophysiological examinations in the same animal. In the present study, tcMEPs could be recorded over a longer period because continuous electrode placement was unnecessary, and the procedure was minimally invasive since the electric needle was only inserted for each recording.

The tcMEPs amplitude in the hindlimbs was lowest on day 1 and then recovered over time to 85.9% of the baseline on day 28. Conversely, the amplitude in the forelimbs did not begin to recover until day 14, and then it improved to 81.3% of the baseline on day 28. A previous report using the cylinder test in cortical and striatal cerebral infarction rat models showed only a slight improvement in forelimb function up to 14 days after onset, and then showed a moderate improvement until day 28 ⁷. Our quantitative evaluation of the forelimb amplitude of tcMEPs was consistent with their qualitative results obtained from the cylinder test. The qualitative mNSS method for assessing locomotor function showed moderate improvement on day 7 and then continuous improvement over time. We found that our quantitative evaluation of the hindlimb amplitude at tcMEPs was consistent with the results of mNSS. We speculated that the delay of forelimb compared to the hindlimb improvement might be caused by the area of MCAO infarction; the forelimb area was at the core, and the hindlimb area was at the marginal section of the infarction. Future studies should explore mechanisms underlying the difference between the improvement of forelimb and hindlimb function with the evaluation of not only mNSS, but also with the cylinder test.

To date, there have been no reports of long-term recordings of tcMEPs in the forelimbs in ischemic stroke models. One reason may be that tcMEPs of the forelimbs are difficult to evoke because the motor area of the forelimb is located more outward than that of the hindlimbs, making it more difficult for the electrical stimulation to be transmitted. Our method of tcMEPs via thinning the skull could reduce electrical resistance and overcome the challenge of electrical transmission. With this method, we were able to evaluate the motor function of not only the hindlimbs but also the forelimbs in an ischemic stroke rat model. Long-term recordings allowed us to evaluate the changes in motor function in detail.

The latency of tcMEPs in both the fore- and hindlimbs did not change significantly over the 28 day follow-up period. The latency of muscle response to cortical stimulation represent the sum of central and peripheral conduction times, and central motor conduction time is calculated by subtracting the peripheral conduction time from the MEPs latency elicited by motor cortical stimulation ²¹. When patients' corticospinal tracts are damaged, axonal degeneration is reported to occur about 1 month after onset, prolonging central motor transmission time ^21,22. In contrast, in rats, axonal degeneration of the corticospinal tract is reported to occur within 1 to 3 weeks after ischemic stroke, but it is unclear whether the central motor conduction time is prolonged as well ²³. In addition, central motor conduction time has been reported to correlate with the severity of neurological defects ²⁴. Therefore, in the present study, the latency may have remained almost unchanged because of motor function improved over time after an ischemic stroke. On the other hand, there is a report that the latency of tcMEPs in both the fore- and hindlimbs was prolonged in rats 7 days after ischemic stroke, which differs from our results ²⁰. However, it has also been reported that the degree of cerebral infarction and cerebral edema affect the central motor conduction time ²⁵, and this difference may be due to differences in the severity of the ischemic stroke models.

Behavior rating scales such as the mNSS have the advantage of being easy to measure, but they are qualitative. In contrast, tcMEPs are quantitative and can more accurately assess the motor function of rats. Furthermore, our results show that forelimb and hindlimb motor functions can be evaluated separately. This minimally invasive and longitudinal quantitative method could be useful for further research, such as studies investigating the effectiveness of cell therapies and rehabilitation in cerebral infarction rat models.

In conclusion, we established a highly reproducible tcMEPs method for the longitudinal assessment of motor function at not only the hindlimbs, but also the forelimbs in a MCAO rat model. We showed the difference in tcMEPs amplitude recovery timing between the forelimbs and the hindlimbs after MCAO. Quantitative assessment of forelimb and hindlimb motor function in rats appears to be essential for understanding the mechanisms of brain damage in stroke.

Ethics statement

All methods have been reported in accordance with the ARRIVE guidelines (https://

arriveguidelines. org) and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). All study protocols were approved by the Animal Testing Committee Guidelines of Hiroshima University, Japan, and conducted under the authority of the Project License (A19-164).

Electrophysiological recordings

Five female adults Sprague–Dawley rats (Charles River, Kanagawa, Japan) with a bodyweight range of 250-300 g were housed at a room temperature of 23 ± 1 °C, humidity of 50 ± 10%, and a light/dark cycle of 12 h with ad libitum access to food and water. Referring to previously published reports that have recorded tcMEPs in a similar number of animals ²⁶, we deemed our sample size sufficient to evaluate our model. Inhalation anesthesia was induced with 1.5% isoflurane, and during the procedure, body temperature was maintained at 37 ± 0.5 °C using a thermostat-controlled heating pad (Bio Research Center Co., Ltd., Nagoya, Aichi, Japan). After the linear skin incision, the skull was exposed, and then the skull was thinly shaved (total area diameter = 5 mm) with a diamond drill at the bregma and lambda positions as previously reported (Figure 5a) ^17,18. This skull thinning technique created a low electrical resistance field for optimal electrical stimulation of the motor cortex. Transcranial electrical stimulation was performed through the thinned skull position using needle electrodes (Natus Medical Inc., Middleton, WI, USA). The anode was installed at the lambda position and the cathode at the bregma position. Transcranial electrical stimulation was performed by administering four stimulus trains over a total duration of 0.5 ms with supramaximal stimulation. The strength of stimulation for each rat was decided before the MCAO procedure, and the stimulation strength was kept constant during the time course of tcMEP experiments for each subject. The mean stimulus intensity of MCAO models for the forelimb and hindlimb was 34 V and 66 V, respectively. To record tcMEPs, two needle electrodes were inserted in the forelimb's extensor muscles and the hindlimb's quadricep muscles, respectively, and the ground wire was inserted into the tail (Figure 5b). Stimulation and recording were performed using Endeavor CR (Nicolet Biomedical, Madison, WI, USA), and the needles for transcranial electrical stimulation were inserted percutaneously before every recording and then removed after every recording. The latency and amplitude of tcMEPs were recorded before and 1, 7, 14, 21, and 28 days after the MCAO procedure.

Surgical procedure for MCAO and the neurological function test

After measuring the reference value of tcMEPs, the transient MCAO procedure was performed by an intraluminal thread occlusion method, as previously described ²⁷. The right common carotid artery, external carotid artery, internal carotid artery, and pterygopalatine artery were exposed under isoflurane anesthesia using a microscope. A 4-0 nylon monofilament suture coated with silicone (403745PK10. Doccol Co., Sharon, MA, USA) was inserted from the cut stump of the external carotid artery toward the internal carotid artery until it blocked the origin of the right MCA. Following one and half hours of occlusion, the thread was retracted for reperfusion. Body temperature was maintained at 37 ± 0.5 °C as described above during the procedure.

Neurological function was assessed using mNSS, which is a composite score of motor, sensory, balance, and reflex responses on a scale from 0, indicating normal, to 18, indicating major impairment ²⁸. The mNSS tests were performed before and on days 1, 7, 14, 21, and 28 after MCAO. The mNSS scores of all rats on day 1 after the MCAO procedure were between 9 and 11, and we used these rats in this study.

Statistical analysis

Continuous variables were presented as the mean (standard deviation) and 95% confidence interval (CI). Weekly continuous variable comparisons were performed using repeated measures one-way analysis of variance (ANOVA). P-values < 0.05 were considered statistically significant. Statistical analyses were performed using JMP^® Pro version 16 (SAS Institute, Cary, NC, USA).

Data Availability Statement

The raw data supporting the findings of this study are displayed in Figs. 1, 2, 3, 4, and 5. The authors declare that all data supporting the findings of this study are available within the paper.

Acknowledgments

The authors acknowledge with thanks the financial support from a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant No. 20K09348 and JSPS KAKENHI Grant No.21K16609)

Authorship contribution statement

All authors contributed to the study conception and design. MH, MT, and YM performed animal experiments. MH, NM, and YM analyzed the data. The project was directed and supervised by FY and MT. The first draft of the manuscript was written by MH, and all authors commented on previous versions of the manuscript. All authors have read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

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Establishment of longitudinal transcranial stimulation motor evoked potentials monitoring of the forelimbs and hindlimbs in an ischemic stroke rat model

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