Upregulation of RAB7 Relates To Neuronal Pyroptosis After Spinal Cord Injury In Rat

Chao Liu The Second A liated Hospital of Soochow University Qianliang Wang The Second A liated Hospital of Soochow University Sunao Li Soochow University Xueshi Chen Soochow University Xinqi Huang Soochow University Jun Yan (  liuchaotougao@163.com ) The Second A liated Hospital of Soochow University https://orcid.org/0000-0003-1970-1600 Haiyan Shan The A liated Suzhou Hospital of Nanjing Medical University Mingyang Zhang Soochow University


Introduction
SCI is a devastating disease, an a iction of the nervous system, results in disturbed sensory, autonomic, and motor functions and a nancial burden (Anjum et al. 2020). SCI may be divided into both primary and secondary mechanisms of injury. During SCI, most of the cells died because of injury-induced biochemical changes (secondary injury). The secondary injury induced by SCI leads to cell death may persist for several days to weeks and cause nervous malfunction (Zhu et al. 2017). Studies have demonstrated that apoptosis, in ammation, tissue ischemia, cell edema, and other pathophysiological converts can greatly reinforce the damage level of SCI which leads to multiple neurological dysfunctions after trauma ( The Rab proteins are one of the Ras small GTPase superfamilies, a large amount of literature has shown that Rab proteins are key regulators of membrane tra cking, cell signaling, cell growth and development by binding to effecter proteins (Gonzalez-Gaitan and Stenmark 2003; Snider 2003). Rab7 has been regarded as an important factor in endosomal membrane tra cking in neuronal cells (Bucci et al. 2000).
Additionally, Rab7 was found localized in the lysosome, and the late endosomal membrane fusion and tra cking were regulated by Rab7 (Chavrier et al. 1990;Zhang et al. 2009).
Studies demonstrated that Rab proteins are related to neurological functions and expressed in glia and neurons, and the mutation or disfunction of Rab7 may cause neurological disorders (Ng and Tang 2008). Cataldo , s reports have shown that the defects of Rab7, which regulates late endocytic tra c, are associated with Down's syndrome and Alzheimer's disease (Cataldo et al. 2008). In neurons, Axonal transport is key for its survival and differentiation and helps to long-range communication of neurons.
Saxena, et al (Saxena et al. 2005)showed that, in the endosome, inhibiting Rab7 activity leads to aggregation of tropomyosin receptor kinase A (TrkA) and potentiated NGF-stimulated signaling of TrkA to advance neurite outgrowth. These data indicate that Rab7 has a crucial position in the central nervous system (CNS). However, the role of Rab7 in spinal cord function has not been investigated. Furthermore, currently, most studies are performed in vitro with cells. The experimental with animal models may have additional insight on the underlying mechanisms of Rab7 in neuropathy.
In our study, we show the distribution and expression of Rab7 in the spinal of rats after SCI, and rst investigate the relation between Rab7 and pyroptosis after SCI. Aimed to gain a deep perception about the Rab7 functions in injured and normal spinal and its roles in the molecular and cellular mechanisms underlying nervous system damage and repair.

Animals and SCI model
Male adult Sprague Dawley (SD) rats weighing 220-250g (6-8 weeks old) were used in this study, and those rats were gained from the Laboratory Animal Center of Soochow University. They are placed with constant temperature and humidity, and a 12-hour light-dark cycle. All procedures about animals were permitted by the Institutional Animal Use and Care Committee at Soochow University and the China Laboratory Animal Guidelines. In experiments, data were obtained by investigators blinded to the study group. In addition, all experiments were conducted during the daytime (7:00-19:00). The most important point is that all efforts were taken to obtain minimal suffering and reduction for animal use.
In the SCI group, the rats were established by using the modi ed weight-drop method as previously described (Dai et al. 2019). All animals were divided into two experimental groups, randomly, with 6 rats per group: Sham group, SCI group (1 h, 6 h, 12 h, 1 d, 2 d, 3 d,7 d). Rats were anesthetized by injecting pentobarbital sodium (50 mg/kg) into the peritoneum. Brie y, The T8-T10 lamina was removed after exposure, and the spinal cord was exposed, fully. Laminectomy was operated at the T9 level. We use a dropping bar which was 9 g and 2.5 mm in diameter to hit the exposed spinal cord from a height of 6.0 cm, The spinal cord hemorrhage, the delayed extension of hind limbs and tail swing indicated the successful establishment of the SCI model. Subsequently, the incision was sutured layer by layer. Signs of successful modeling included spinal cord hemorrhage, convulsions in hind limbs and tail swing. Until spontaneous urination was resolved, the bladder of rats was evacuated manually. The spinal cord specimens were obtained for the follow-up experiments.

Western Blot Analysis
An overdose of chloralhydrate was used to treat rats and then be killed at different points (n = 6); the spinal cords (1cm) were removed and stored at -80℃ until further use. Proteins were distilled from spinal cord tissue in RIPA lysis buffer. Then use an enhanced BCA protein assay kit to determine the concentrations of protein. Equal quantities of protein were divided via sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene di uoride (PVDF) membranes. and the PVDF was then blocked with 5% BSA for 2 h at room temperature, and then the PVDF was incubated with the relative primary antibodies overnight at 4°C. The primary antibodies were as follows: Anti-Rab7(1:400), Anti-GSDMD (1:500), Anti-Caspase 1 (1:100), and Anti-ASC (1:200) antibodies were acquired from Santa Cruz Biotechnology. After washing by TBST, blots were incubated with secondary antibodies at normal temperature for 2 h. the ECL chemiluminescence system was used to measure the blots and then captured on autoradiographic lms. The lms were then scanned. The densitometric analysis of the bands was conducted with Image J software.

Sections and Immunohistochemistry
The chloral hydrate (10% solution) was used to anesthetize rats, terminally. Then the rats were perfused with 50mL of 0.01M PBS, pericardially, and nally used 4% paraformaldehyde. The spinal cord was removed after perfusion and post xed in the xative for 12 h, and then 20 % and 30 % sucrose for 1 day, respectively. An optimal cutting temperature compound was used to embed the spinal cords. 5-μm frozen cross-sections were prepared for the examination. The slices can be frozen in a refrigerator at -80℃. The sections were blocked with bovine serum albumin (BSA) for 2 h at normal temperature. The anti-Rab7 antibody (anti-mouse, 1:100) was used to incubate the sections overnight at 4 °C, then the sections were incubated in a related secondary antibody (Vector Laboratories, Burlingame, CA, USA). The DAB (Vector Laboratories) was used to visualize staining, The strong or moderate brown staining of cells was regarded as positive, cells with weak staining were scored separately, while cells were regarded as negative when with no staining.

Double Immuno uorescent Staining
To avoid the sections unspeci c staining, 10 % normal serum blocking solution which contains BSA and Triton X-100, and Tween-20 was used to block at room temperature for 2 h, and the sections were then incubated with primary antibodies for anti-ASC (1:50; Abcam; rabbit), antibodies for anti-Rab7 (1:50; Santa Cruz; mouse), anti-NeuN(1:500; Sigma), anti-GFAP(1:500; Sigma), anti-IBA1(1:500; Sigma). Above all, the primary antibodies were used to incubate the sections overnight at 4 °C, then a mixture of uorescein isothiocyanate and tetramethylrhodamine of secondary antibodies were used to incubate at room temperature for 2 h. Leica Spectral Confocal microscope (Germany) was used to examine the stained sections.

Statistical Analysis
Statistical analyses were dealt with GraphPad8.0. Results are presented as means ± SEM. The statistical was determined with one-way analysis of variance (ANOVA) followed by Student t-test (two means comparison) or Tukey's post hoc multiple comparison tests. P<0.05 was considered signi cant.

Result
The Rab7 Expression Following SCI To identify the Rab7 expression pattern, the expression of Rab7 protein was examined by Western blot at different time points after SCI and in sham control. In the spinal cord around the lesion site, Rab7 protein markedly increased on the 24 h compared to sham groups, then decreased at 48 h, and attained a peak on day 3 (Fig. 1).

The Rab7 Immunoreactivity Staining In Rat After Sci
To recognize the Rab7 immunoreactivity changes in medulla spinalis three days after spinal cord injury, an anti-Rab7 mouse monoclonal antibody was used to perform the immunohistochemistry for transverse cryosections of myeloid tissue, 5 mm around to the damaged site. Markedly, Rab7 staining was expressed in the myeloid tissue (Fig. 2). In addition, Rab7 expression was estimated by counting cells between sham and spinal cord injury groups (Fig. 2); the results were comparable with Western blot (Fig.   1).

Expression of the Neuronal Pyroptosis In Spinal Cord After Injured
To con rm the links between Rab7 and pyroptosis cells, the studies were performed as follows: The level of pyroptosis-related proteins (ASC, Caspase-1, GSDMD) in myeloid tissue was detected by immunoblotting. Those proteins activation occurred in the myeloid tissue of rats after SCI with outstanding activation occurring 24 h post-injury, and reaching the peak at day 3 after SCI (Fig. 3). To reveal the pyroptosis cell types after SCI, the double immuno uorescence staining was with various phenotype-speci c markers and ASC-speci c antibodies (Fig. 5). On the third day, most of the reactive neurons were ASC-positive (Fig. 5). The Rab7-expressing neurons were partly revealed as ASC positive (Fig. 4). And, double immuno uorescent staining indicated that Rab7 was colocalized with ASC (Fig. 6).

Discussion
To reveal the distribution and expression of Rab7 in the medulla spinalis of rats after injury, we established the animals' SCI models. In our experiment, we elucidated the upregulation of Rab7 in rat medulla spinalis after SCI. Rab7 was obviously observed in the rat spinal cord and speci cally expressed in the neurons of the spinal cord, however, the detectable levels of Rab7 were not be observed in astrocytes and microglia. The immunohistochemistry demonstrated that Rab7 is strongly expressed in the SCI group compared with the sham group. Furthermore, immunoblot analysis demonstrated that the protein level of Rab7 gets to a peak on day 3 after SCI. Surprisingly, we also observed that pyroptosis cells marker ASC speci cally expressed in the neurons of the spinal cord and there was also peak accumulation of ASC at 3 days post-injury. To con rm the links between Rab7 and pyroptosis cells, we performed double immuno uorescence staining with Rab7 and ASC. The result revealed that Rab7expressing neurons were partly shown as ASC positive. In addition, uorescence microscopic immunolabeling of ASC in spinal sections demonstrated that neuronal undergo pyroptosis after SCI. To sum up, it showed that in the traumatic spinal cord, the expression of Rab7 may be induced in the preoptic neurons after SCI. These data may provide signi cant insights to understanding the cellular and molecular mechanisms underlying SCI.
It was reported that Rab GTPases was the largest family of small GTP-binding proteins that contains more than 60 members in the human genome . Rab7 is one of this large family and has a unique role in modulating autophagy (Wen et al. 2017). Autophagy is a catabolic process via degrading protein aggregates, cytoplasmic proteins and organelles, and is tightly correlated to cellular metabolism Pyroptosis, the other form of programmed necrosis, is a pro-in ammatory form of regulated cell death that depends on the enzymatic activity of in ammatory proteases that pertain to the cysteine-dependent aspartate-speci c protease (caspase) family. Pyroptosis is mainly observed in professional phagocytes of the myeloid lineage, The higher levels of expression of in ammatory caspases of these cell types may be the possible mechanism. However, it has also been observed in neurons and other cells. The occurrence of pyroptosis depends on the activation of the in ammasome which leads to the recruitment of the adaptor protein ASC. In many studies, ASC can be one of the reliable markers for pyroptosis.
Previous studies indicated that pyroptosis markers increased in the spinal after injury Liu et al. 2020;Wu et al. 2020). In our experiment, the effect of SCI on regional cellular patterns of expression of ASC was detected. These results are consistent with ASC activation that appeared in the spinal cord after SCI. However, the timing sequence of pyroptosis in SCI has not previously been reported incomparable detail. In our study, on the rst day, the pyroptosis-related proteins signi cantly increased and then attained a peak on day 3 after SCI. Interestingly, those proteins then decreased at 48 h, This might be related to the mechanisms mediating the autoimmune response after SCI. Studies demonstrated that the vascular immediate disrupt after injury and then trigger secondary pathology, the immune cells extravasated around the lesion site conduct pressure on the damaged spinal tissues and destroy the blood ow, fourthly, this state continues up to 24 h (Alizadeh et al. 2019). Due to different injuries in the spinal cord injury animal model, the timing sequence of pyroptosis in SCI remains inconclusive. Therefore, it will be of great value to conduct an in-depth exploration of it.
Evidence showed that pyroptosis played an important function in the pathogenesis of multiple neurological disorders as well as SCI (Al Mamun et al. 2021). Compared with the changes of pyroptosisrelated proteins after SCI, Rab7 was also region-speci c and time-dependent. the neurons of Rab7 expressing partly showed as well as ASC positive. Thus, we hypothesized that upregulation of Rab7 in the spinal cord was strong links to the pyroptosis-related proteins after SCI, suggesting a role that Rab7 may play a critical function in pyroptosis after SCI Above all, the evidence indicated that Rab7 has an important function in the process of pyroptosis induced by SCI. We rst showed the expression pro le of Rab7 in rat spinal cord after SCI. Our results may provide signi cant insights to understand the underlying cellular and molecular mechanisms of CNS after SCI and a new strategy for the treatment of SCI. The function of Rab7 on cellular pyroptosis, however, still needs further research, and future studies also attempt to explore their possible substrates and downstream signaling targets.

Declarations Con icts of Interest
The authors declare no con ict of interest.

Authors' Contributions
Chao Liu and Qianliang Wang contributed equally to this work. Figure 1 The Rab7 expression changes after SCI.

Figures
(a)spinal cords of rats were extracted at different times after SCI for analysis. Immunoblots probed for Rab7 and GAPDH are shown. The bar chart below indicates the ratio of Rab7 relative to GAPDH. Rab7 protein increased at 24 h compared with sham groups, signi cantly, and attained a peak on day 3, and decreased at 2 days. (b) Semiquantitative analysis of the intensity of staining of Rab7 to GAPDH. The data are means ± SEM (n=3, ** P<0.01, signi cantly different from the sham group).

Figure 2
Immunohistochemistry expression of Rab7 in the spinal cord surrounding the wound after SCI.
Higher-power views of sections immunostained with antibody for Rab7 in the spinal cord of sham group  Sample immunoblots probed for pyroptosis-related proteins (ASC, Caspase-1, GSDMD) is shown above.
The proteins expression was increased at 24 h after injury and it attained a peak level at day 3 after SCI and decreased at 2 days. Quanti cation graphs (relative optical density) of the intensity of staining of pyroptosis-related proteins (Caspase-1, ASC, GSDMD) to GAPDH. GAPDH was compared to con rm an equal amount of protein was run on the gel. The data are means ± SEM (n = 3, * P<0.05, ** P<0.01, *** P<0.01, signi cantly different from the sham group).

Figure 4
Double immuno uorescence staining for Rab7 and cell marker (NeuN, GFAP, IBA1) in the spinal cord at day 3 after SCI The spinal cord of rats within 5 mm distance around the injury site on the third day after SCI, sections labeled with Rab7 (red) and cell marker (NeuN, IBA1, GFAP) (green). Dapi was used to counter-stained with nuclei (blue). In the merged images, the yellow color represented the colocalization of Rab7 with NeuN. Double immuno uorescent staining showed that Rab7, compared with the sham group, was signi cantly increased in the spinal cord at 3d after injury, and Rab7 and IBA1 or GFAP were not colocalized. Scale bars 50μm.

Figure 5
Double immuno uorescence staining for ASC and cell marker (IBA1, GFAP, NeuN) in the spinal cord Sections were labeled with cell markers (NeuN, GFAP, IBA1) (green) and ASC (red). Dapi was used to counter-stained with nuclei (blue). In the merged images, the yellow color represented the colocalization of ASC with NeuN. Double immuno uorescent staining showed that ASC, compared with the sham group, was signi cantly increased at 3d after injury. ASC and GFAP or IBA1 were not colocalized. Scale bars 50μm.

Figure 6
Double immuno uorescence staining for ASC and Rab7 in the spinal cord The rats , spinal cord within 5 mm distance around the injury site on day 3 after SCI, sections were labeled with ASC (green) and Rab7 (red). Dapi was used to counter-stained with nuclei (blue). In the merged images, the yellow color visualized represented the colocalization of ASC with Rab7. Double immuno uorescent staining demonstrated that ASC was colocalized with Rab7. Scale bars 50μm.