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    L4-to-L4 nerve root transfer for hindlimb hemiplegia after hypertensive intracerebral hemorrhage
  • Figure 1|Diagram of establishment of rat models and treatments for each group. 

    Using published nerve tracings as a guide (Frost et al., 2013; Song et al., 2017), we created a hematoma-caused lesion in the posteromedial area of the PLIC. Rats were anesthetized with 2% pentobarbital sodium intraperitoneally with body temperature maintained at 37 ± 0.5°C using a heating pad. Briefly, after rats were restrained in a stereotactic frame (RWD-68025, Shengzheng Biotechnology Co., Ltd., Nanjing, Jiangsu Province, China), a scalp incision was made along the midline, hemostasis was performed using a bipolar electrocoagulator (Johnson & Johnson Medical Devices Co., Ltd., Suzhou, Jiangsu Province, China), and then a small hole was drilled on the skull. Subsequently, a hydraulic microinjector was gradually inserted perpendicularly into the target area (anteroposterior, ?3.1 from bregma; mediolateral, ±3.3 from the midline; dorsoventral, ?7.8; n = 30) (Figure 1A and B). Approximately 180 μL blood or saline was slowly injected into the target site for 15 minutes at a rate of 12 μL/min through a 30G Hamilton syringe connected to an UltraMicroPump (WPI, Sarasota, FL, USA). The sham-operation group received a saline injection, followed by exposure of only bilateral L4 nerve roots. After surgery, all rats were transported to a recovery chamber with ketoprofen (2 mg/kg, 
    i.m.) for analgesia for 3 consecutive days. Seven days after injection, T2-weighted magnetic resonance imaging (MRI) was used to locate the affected area (Figure 1C). Both behavioral and electromyographic tests confirmed the establishment of the rat model. 
    A beam-walking test, ladder rung walking task, and footprint analysis were performed as per the established protocols at baseline, 9, 13, and 17 weeks post-operation (Kemp et al., 2010). At 15 days before the baseline valuations, rats were trained on each procedure. A schematic illustration of the corresponding protocol is shown in Figure 1D

    At 2 weeks after establishment of the rat hemiplegia models, L4-to-L4 nerve root transfer was performed in the L4-L4 transfer group, bilateral L4 nerve root transfection was conducted in the Bi-L4 transection group, and exposure of bilateral L4 nerve root only was performed in the sham-operation group. After ether inhalation and anesthesia by an intraperitoneal injection of 2% pentobarbital sodium, rats were laid in supine position, shaved, and fixed on the miniature operation table. A median incision (3–4 cm long) was made longitudinally in the abdomen, which was centered on the L4 and paralleled the anterior superior iliac spine. Bilateral L4 nerve roots were observed under the operating microscope (SZ61, Olympus, Tokyo, Japan). For the L4-to-L4 root transfer, the left L4 root (intact side) was traced and transected as distally to the intervertebral foramen as possible, whereas the right L4 root was severed as proximally as possible. The proximal stump of the left L4 nerve root was transferred to the distal stump of the right L4 root using 10-0 Prolene sutures, with the right proximal stump secured to the ambient psoas major. In the bi-L4 transection group, the stumps of bilateral L4 nerve roots were fixed to the muscle to avoid neural reconnection (Figure 1E and F). An absorbable hemostatic sponge was carefully stuffed around the operative field before strict skin closure. 


    Figure 2|Behavioral and electromyographic assessments confirming establishment of the rat model. 

    At 7 days after injury to the dorsomedial area of PLIC, rats began to exhibit reduced activity in the right lower extremity and galloped in circles centered on the right hindlimb. In the walking tasks, there was a > 85% slip rate in the right hindlimb compared with the left hindlimb (Figure 2A and B). In the footprint analysis, the forepaw and hindpaw prints on the paretic side were non-overlapping for at least 6 months, with a spacing of 19.2 ± 0.7 mm  (Figure 2D). In contrast, the spacing on the intact side had a very small interval 1.2 ± 0.1 mm (Figure 2C). 
    Additionally, denervated MUAPs in the hemiplegic hindlimb had a lengthened duration and shortened amplitude (Figure 2F) compared with those in the left hindlimb (Figure 2E), which is consistent with the results of the footprint analysis. The results imply a successful establishment of a rat model of hemiplegia after HICH. 


    Figure 3|Availability of L4 as a source nerve. 

    Anatomically, a nerve root that controls flexors and extensors is usually considered as an optimal donor candidate. We performed initial experiments to screen a donor nerve. At 10 days after transection of L4 only, acupuncture electromyography showed that myokymic potentials and positive sharp waves were prominently detected in the quadriceps femoris, moderately detected in the semimembranosus and gastrocnemius, slightly detected in the tibialis anterior, and hardly detected in the biceps femoris (Figure 3A). In contrast, in the condition of retention of the L4 nerve root and excision of other lumbar roots, normal potentials were observed in the corresponding muscles, particularly in the quadriceps femoris, semimembranosus, gastrocnemius, and tibialis anterior (Figure 3B). The maximum BBB scores were 20 and 13 for transection of the right L4 nerve root only and retention of the right L4 only, respectively (Figure 3C). The BBB scores suggested that under the condition of retention of the L4 nerve root only, the hindlimb function might recover to approximately 70% of a na?ve state within 2 weeks. Electromyography indicated that the muscles controlled by L4 could perform flexion and extension for major joints in the hindlimb, especially for the ankle. These results demonstrated eligibility of the L4 nerve root as a source nerve. 


    Figure 4|Behavioral improvements following the L4-to-L4 transfer. 

    The right forepaw and hindpaw prints from the footprint analysis of a na?ve rat and model rat are shown in Figure 4A, and the intervals between the forepaw and hindpaw prints on the affected side are shown in Table 1. In the L4-L4 transfer group, the interval between the forepaw and hindpaw prints during L4-to-L4 root transfer significantly diminished over time until an overlapping status emerged at 17 weeks after surgery (Figure 4B). In the beam-walking test and ladder rung walking task, the overall slip rate for the bilateral hindlimbs did not statistically differ between the bilateral (Bi)-L4 transection and L4-L4 transfer groups and attained 90% at baseline (Figure 4C and D).

    At 1 week, the slip rate in the Bi-L4 transection group drastically declined to 44–56% as compared with that of the L4-L4 transfer group. At 3 weeks, rats in the sham-operation group achieved approximately 95% accuracy, in contrast to approximately 57% accuracy in the Bi-L4 transection group and approximately 13% accuracy in the L4-L4 transfer group. At 5 weeks, in the Bi-L4 transection and L4-L4 transfer groups, behavioral improvement was observed, with greater improvement observed in the Bi-L4 transection group. From 9 weeks onward, rats in the L4-L4 transfer group displayed greater reductions in the slip rate than did those in the Bi-L4 transection group. Although an increase in accuracy was observed after 9 weeks, rats in the Bi-L4 transection and L4-L4 transfer groups did not improve to the level of the sham-operation group at any time point. The BBB score in the L4-L4 transfer group was significantly higher at 17 weeks than at 9 and 13 weeks (Figure 4E). The data suggest effective motor function recovery of the hemiplegic hindlimb after L4-to-L4 root transfer.


    Figure 5| Electromyographic assessment demonstrating reinnervation of the denervated muscles. 

    In the L4-to-L4 root transfer group, the reinnervated MUAPs could be elicited with concentric needles in the quadriceps femoris, semimembranosus, lateral gastrocnemius and tibialis anterior of the right hindlimb in 4 out of 10 rats (40%) at 9 weeks, in 7 rats (70%) at 13 weeks, and in 9 rats (90%) at 17 weeks after surgery (Figure 5A and B). Also, the denervated MUAPs detected in the target muscles markedly decreased over time. In H-reflex recordings, the M/H-wave latency for the right gastrocnemius was remarkably longer at 9 weeks than that at 17 weeks (P < 0.05) (Table 2 and Figure 5C). The latency shortened over time until it reached an approximately normal level (P > 0.05). Together, the results suggested that the L4-to-L4 nerve root transfer efficiently reinnervated the targeted end-organs (Figure 5). 


    Figure 6|Retrograde tracing showed fluoro-gold-labeled motoneuron number increased over time. 

    In the ventral horn, many fluorogold-labeled motoneurons were observed (Figure 6A). At 9, 13, and 17 weeks (Figure 6B–E), the number of labeled motoneurons increased over time, in contrast to the contralateral side at the same time points (P < 0.05 for each time point). These results showed that an increasing number of regenerated axons reinnervated the motor endplates. 


    Figure 7| Immunofluorescence showed axon regeneration. 

    Figure 8| Ultrastructure demonstrating axon regrowth and remyelination.  

    At the coaptation site, regrowing axons were observed, with MAP-2-labeled axons accounting for 25.61 ± 5.18% at 9 weeks, 38.52 ± 3.64% at 13 weeks, and 57.89 ± 6.25% at 17 weeks (Figure 7) of all axons. Additionally, reinnervation between the stumps increased at each time point until reaching nearly complete reinnervation at 17 weeks (P > 0.05). Myelinated and unmyelinated axons were observed with ultrastructural analysis, further indicating progressing axon regeneration over the three time points (P < 0.05) (Figure 8). Over time, the G-ratio for regrowing axons declined, in parallel with an elevation of myelinogenesis. Additionally, more reinnervated axons were observed in a realigned and rearranged manner. The results demonstrated effective regeneration of nerve fibers. 


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  • 发布日期: 2021-12-21  浏览: 405
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