Photobiomodulation augments the effects of mitochondrial transplantation in the treatment of spinal cord injury in rats by facilitating mitochondrial transfer to neurons via Connexin 36

Abstract Mitochondrial transplantation is a promising treatment for spinal cord injury (SCI), but it has the disadvantage of low efficiency of mitochondrial transfer to targeted cells. Here, we demonstrated that Photobiomodulation (PBM) could promote the transfer process, thus augmenting the therapeutic effect of mitochondrial transplantation. In vivo experiments, motor function recovery, tissue repair, and neuronal apoptosis were evaluated in different treatment groups. Under the premise of mitochondrial transplantation, the expression of Connex36 (Cx36), the trend of mitochondria transferred to neurons, and its downstream effects, such as ATP production and antioxidant capacity, were evaluated after PBM intervention. In in vitro experiments, dorsal root ganglia (DRG) were cotreated with PBM and 18β‐GA (a Cx36 inhibitor). In vivo experiments showed that PBM combined with mitochondrial transplantation could increase ATP production and reduce oxidative stress and neuronal apoptosis levels, thereby promoting tissue repair and motor function recovery. In vitro experiments further verified that Cx36 mediated the transfer of mitochondria into neurons. PBM could facilitate this progress via Cx36 both in vivo and in vitro. The present study reports a potential method of using PBM to facilitate the transfer of mitochondria to neurons for the treatment of SCI.


| INTRODUCTION
Spinal cord injury (SCI), as a kind of disability disease with a high incidence worldwide, often leads to the impairment of motor and sensory functions in patients. At present, its treatment is still a thorny problem. As the "powerhouse" of cells, mitochondria participate in various fundamental biological processes and its damage has been considered an important cause of neuronal injury in SCI. [1][2][3] Neurons primarily rely on mitochondrial oxidative phosphorylation (OXPHOS) to meet their high energy requirements. 4 Adenosine triphosphate (ATP) synthesis in mitochondria is crucial for membrane potential maintenance, nerve conduction, and synaptic plasticity. 5 In addition, axonal injury triggers acute stress, which reduces mitochondrial transportation to the growth cones of neurons for sprouting, thereby causing axonal energy deficiency and axonal regeneration failure. 6,7 There is mounting evidence that directly supplementing mitochondria in the damaged area, which is called mitochondrial transplantation, has become an attractive treatment strategy. Compared with the SCI group, the mitochondrial treatment group partially improved motor function recovery, and histological repair and reduced the inflammatory response and neuronal apoptosis. [8][9][10] It is noteworthy that this strategy still encounters two main problems. First, what is the most suitable source of exogenous mitochondria for transplantation? Autologous liver, heart, and muscle tissues are rich in mitochondria. However, the extraction of these mitochondria results in tissue injury and can only be performed once, limiting the application of this approach. 11 Cell lines such as stem cells and PC-12 cells are the common sources of transplantation, but ethical issues should be carefully considered before clinical practice. 12 Second, how to efficiently facilitate exogenous mitochondria transfer, particularly to neurons. Even in the case of mitochondrial transplantation, the proportion of exogenous mitochondria entering neurons was still not high, let alone the proportion of endogenous astrocyte mitochondria transferred to neurons in the absence of exogenous mitochondria. 11,13 Moreover, macrophages, microglia, and astrocytes can also ingest mitochondria but whether they play a beneficial role after SCI is still unclear. 9,[14][15][16][17] For the first problem, platelet-derived mitochondria are the suitable candidates. The content of platelets in peripheral blood is high, mitochondria could be isolated many times, and the trauma caused by simple venipuncture is less than that caused by muscle mitochondria separation. More importantly, there are no issues related to ethics or immune exclusion, so this treatment strategy has broad prospects for clinical application. 11 For the second problem, we focused on the particularity mechanism of mitochondrial transfer to neuron-gap junctional intercellular communication (GJIC). It is formed by the connexin of a donor cell and a recipient cell that allows ions, signaling molecules, and metabolic molecules to move in and out of cells. 18 The connexin from a single cell can also form a hemichannel and mediate the exchange of materials between the cell and the extracellular environment. 19 In an in vitro study, inhibition of Connexin 43 could reduce mitochondrial metastasis to VSC4.1 motor neurons, while activation of Connexin 43 increases mitochondrial metastasis. 9 However, VSC4.1 motor neurons are different from primary neurons, so further research is needed to demonstrate the specific pathway by which mitochondria enter neurons. Connexin 36 (Cx36) is a specific connexin that only expresses on the surface of neurons and primary neuron-dorsal root ganglia (DRG) but not on other cells, such as astrocytes and macrophages. [20][21][22] Therefore, we speculated that Cx36 could be the key point of mitochondrial transfer to neurons and DRGs which might provide a novel insight into the treatment of SCI.
Studies have shown that prolonged dark adaptation decreased the expression of Cx36 in the retina while increasing laser exposure promoted Cx36 expression. 23,24 Photobiomodulation (PBM), also called lower-level laser therapy, has attracted much attention in the past 10 years. Its main principle is that cytochrome c oxidase absorbs photons of near-infrared laser to promote mitochondrial bioenergetic and final ATP production. 25 In addition, PBM could regulate reactive oxygen species (ROS) production and activate a variety of transcription factors thus it has been used to treat SCI, ischemic stroke, traumatic brain injury, Alzheimer's disease and so on. [26][27][28][29] However, mitochondrial dysfunction that occurs after SCI limits the therapeutic effect of PBM. 30 To address the problems mentioned above, we first studied embedded laser fiber-mediated PBM combined with platelet-derived mitochondrial transplantation in the treatment of SCI. Our results indicated that combined treatment was better than that of a single treatment in terms of motor function recovery, tissue repair, and inhibition of neuronal apoptosis. Next, we found PBM enhanced the expression of Cx36 in the spinal cord and promoted the transfer of mitochondria into neurons, as well as the specific downstream effects such as ATP production and antioxidant capacity. Finally, we verified our speculation by using an inhibitor of Cx36 on DRGs in vitro.

| Platelet-derived mitochondrial isolation and analysis by flow cytometry
The protocol for platelet and mitochondrial isolation was performed as previously described. 31 Ten milliliters of rat blood were centrifuged twice at room temperature for 5 min each to take out cell debris. The supernatant was centrifuged at 800Âg for 10 min to collect a white pellet, that is, platelet granules. Then, 5 ml of prechilled GENMED lysis working solution (GMS10062.2, GenMed Scientifics, Inc., USA) was added, and the slurry was homogenized. The samples were centrifuged at 4 C for 10 min at a speed of 1500Âg to remove the remained cells. The supernatant was aspirated and centrifuged at 4 C for 10 min at a speed of 11,000Âg to obtain mitochondria. A mitochondrial storage solution (C3609, Beyotime, China) was added to resuspend the mitochondria. Platelet purity as determined using flow cytometry (Beckman F500, USA): APC anti-mouse/rat CD42d antibody (148,505, BIOLEGEND, USA) was added. Samples without antibodies were used as negative controls, and parameters such as the gating strategy and voltage were set using the negative controls. 32 Mitochondria count: mitochondria were pre-stained with Mito Tracker red CMXRos (M7512, Invitrogen, USA) and then mixed with green fluorescent microspheres. Unstained mitochondria and microspheres only were used as negative controls to set the gating strategy and voltage. 31 The specific results are shown in Supplementary is an uncoupler of mitochondrial oxidative phosphorylation that led to the loss of the mitochondrial membrane potential. The control group was treated with 50 μM CCCP (C6700, Solarbio, China) for 20 min, and JC-1 fluorescence was detected. The rats were randomly divided into six groups: the sham group, SCI group, SCI+Vehicle group, SCI+Mito group, SCI+PBM group, and SCI+Mito+PBM group. A modified bilateral spinal cord clamping technique was used to model SCI as previously described. 33 The lamina of T10 was removed to expose the spinal cord. SCI was induced by clamping the T10 spinal cord with forceps for 40 s. The sham group only opened the lamina without clamping the spinal cord. The criteria for successful modeling: rats performed rapid retraction like shaking of the whole body, rapid edema and congestion of the local spinal cord surface, and the dura mater remaining intact.

| Mitochondrial injection
The SCI+Mito group was injected with 10 μl of mitochondria in mitochondrial storage solution using a micro syringe and stereotaxic device as in the previous study. 9 According to the research of others, 9,34 we conducted a pre-experiment and determined that 3 Â 10 5 mitochondria per individual are the best dose. The Mito group in the main body of this article specifically refers to 3 Â 10 5 mitochondria per individual group. The SCI group was injected with 10 μl of mitochondrial storage solution.

| Photobiomodulation therapy
Laser fibers were embedded in the SCI+Vehicle group, SCI+PBM group, and SCI+Mito+PBM group as in the previous study 33 : the front and rear end of the fiber is stitched sequentially on the T8 and T12 spinous protrusions to ensure that the laser could reach T10 spinal cord to carry out PBM. Studies have shown that a laser with a wavelength between 700 and 770 nm has a weak stimulating effect on organisms. A laser with a wavelength >1200 nm is significantly absorbed by water molecules, and a laser with a wavelength of approximately 810 nm has the best therapeutic effect. 35,36 Metaanalysis results suggest that 14 days of PBM could better restore motor function after SCI. 37 Therefore, we choose an 810 nm laser to carry out PBM for 14 days. PBM was performed once a day for 60 min. Rats were irradiated using an 810 nm semiconductor laser

| Gait analysis
For gait analysis, the soles of the hindlimbs were dyed with blue ink, and the dorsal part of the hindlimb was dyed with red ink. Then, the animals were allowed to freely walk down a runway (20 cm wide and 50 cm long) covered with white paper, and researchers calculated the stride length and stride width according to previous methods and definitions. 9,38

| Tissue preparation
Rats were infused with 4% paraformaldehyde (PFA) through the heart, and an approximately 2 cm long piece centered on the site of the injury was obtained. The tissue was embedded in an optimal cutting temperature compound, cut into continuous sagittal or horizontal slices at a thickness of 7 μm, and stored at À20 C.

| Nissl staining
Tissue sections were treated with toluidine blue staining solution (G1032, Servicebio, China) for 5 min. Then, they were treated with 1% glacial acetic acid, incubated in xylene for 10 min, and sealed with neutral gum. The number of neuronal cell bodies at high magnification was determined by Image J software.

| Luxol fast blue (LFB) staining
Tissue slices were placed in myelin staining solution A (G1030, Servicebio, China) for 1 h, immersed in myelin dye B for slight differentiation for 2 s, immersed in myelin dye C for 15 s, and washed until the myelin sheaths were blue and the other components were almost colorless. Then, the sections were dehydrated with absolute ethanol and sealed. The percentage of blue area at high magnification was calculated with Image J software.

| Immunofluorescence
The slices were treated with 0.3% Triton X-100 for 20 min and were blocked with serum for 1 h. Then the slices were incubated with primary antibodies overnight at 4 C. The following primary antibodies

| TUNEL staining
TUNEL staining solution was prepared using a TUNEL kit (C1090, Beyotime, China). The slices were incubated with a secondary antibody and TUNEL staining solution for 1 h. The ratio of TUNEL positive neurons was calculated by Image J.

| Image analysis and quantification
For each rat, three discrete slices were selected near the central canal of the spinal cord with intervals of 100 μm. Pictures were collected in the bilateral areas 200 μm rostral and caudal to the lesion site. Based on the previous analysis method of our group, empirical methods are used for calibration to ensure that unbiased data are collected. 33,39,40 Cell counts in each section were averaged from five independent fields randomly selected within the symmetric cephalad and caudal sides of the lesion center for analysis. All analytical quantifications were performed by independent experimenters.

| Western blotting
The rats were perfused with normal saline, and a 1 cm piece of spinal cord tissue centered on the site of the injury was collected, homogenized, and lysed in the RIPA lysis solution. The supernatant was collected, the bicinchoninic acid (BCA) method was used to determine the protein concentration, and the proteins were boiled, loaded, separated by electrophoresis, and transferred onto a membrane. The membrane was blocked in skimmed milk powder and incubated with the appropriate primary antibody (see Supplemental Table 2) at 4 C overnight. The membrane was incubated with a secondary antibody (1:2000) at room temperature for 1 h, and developed by enhanced chemiluminescence.

| Transmission electron microscopy (TEM)
After the tissue was fixed by glutaraldehyde and 1% osmium tetroxide, alcohol was dehydrated. Embedded with EPON812 resin, made into slices, and then stained for observation. The sections were examined with a HITACHI transmission electron microscope. G-ratio refers to the ratio of the inner diameter to the outer diameter of each axon fiber in about the same 150 axons of five rats in each group and it was calculated by Image J. 41

| Primary neuronal culture
DRG was obtained from SD rat neonates (P0-P2) according to the preliminary research of our group. 42 Briefly, microforceps were used to separate bilateral DRGs under a microscope. Then it was shredded, and trypsin (0.25%, Beyotime, China) and collagenase IV (0.2%, Sigma, USA) were added for digestion for 1 h. DRG neurons were resuspended in a neurobasal medium (Gibico, USA) containing B27, Glutamine, and penicillin/streptomycin. The medium was changed at 24 h and then carried out the next step. After OGD ended, changed back to a normal medium and incubated under normal conditions for 24 h until the next progress. To explore whether connexin mediates the transfer of mitochondria into neurons, we dissolved the connexin inhibitor 18β Glycyrrhetic acid (18β-GA) in DMSO as previously described. 43 DRG cells were preincubated with 18-β-GA (50 μM, SE8280, Solarbio, China) for 8 h and then cocultured for 24 h with exogenous mitochondria. 9 To promote connexin function, the cells were exposed to laser twice per day for 10 min at 9 a.m. and 9 p.m. The growth of neurites was measured with Image J software and the plug-in Neuron J according to the previous methods. 27

| Statistical analysis
All experiments were repeated at least three times independently, and all data are expressed as the mean ± SD. GraphPad Prism 8 software      The above results proved that the therapeutic effect of combined treatment was better than that of a single treatment, hence we began to explore the potential mechanisms of combination therapy. We explored Cx36, which might mediate mitochondrial transfer into neurons and some proteins related to mitochondrial transportation to axons after mitochondrial transplantation. KIF5B, TRAK1/2, and RhoT1/2 GTPases could cooperate directly to facilitate mitochondrial transportation and then promote axon growth. 45,46 In addition, the anchoring protein syntaphilin (SNPH) inhibits mitochondrial movement and then hinders axonal regeneration. 7,47 The results indicated that Cx36 expression achieved a peak at 7 dpi in the SCI+Mito+PBM group but reached a transient peak at Immunofluorescence results showed that PBM treatment could significantly increase the Cx36 level of neurons (MAP2, green) at 3 and 7 dpi (Figure 3b). This trend was consistent with the results of Western blotting of Cx36 (Figure 3a). 3.6 | Combined therapy enhanced mitochondrial function after SCI at 14 dpi

| Inhibition of Cx36 reduced PBM-promoted transfer of mitochondria to DRGs in vitro
Finally, we further validate the effect of PBM on mitochondrial transfer in vitro. As shown in Figure 7a, b, the ratio of mitochondria that entered  show that mitochondrial transplantation alone was not effective enough. 34 In the past several decades, many researchers have tried to improve the efficiency of mitochondrial transfer to cells. The complex formed by the cell-penetrating peptide pep-1 and mitochondria could enhance the efficiency of cellular uptake of mitochondria in Parkinson's disease. In vivo, after intracerebral injection, the ratio of cells containing the labeled peptide was higher than that of unlabeled cells, and the neuroprotective effect of the labeled group was better. 56 However, cell-penetrating peptides have not been used in the clinic.
Recent studies have shown that a stem cell delivery vector promoted the expression of connexin and could improve the efficiency of mitochondrial transfer to damaged cells. 57 But there are still many ethical problems related to the clinical use of stem cells. The optical fiber developed by our research group has been successfully applied in SCI patients and it was proven to be safe and efficinetly. 58 Therefore, we began to explore whether PBM could expand the therapeutic effect of single mitochondrial transplantation and its mechanism.
First, we clarified that the combined therapy had an obvious effect on motor function recovery, tissue repair (Figure 1), and the reduction in neuronal apoptosis ( Figure 2). Second, we found that after PBM intervention, the trend of exogenous mitochondria entering neurons (Figure 4) was consistent with the expression trend of Cx36 ( Figure 3). Notably, Tom 20 ( Figure 5) and TEM ( Figure 5) analysis showed that there was no difference in the ratio of functional mitochondria in the two single treatment groups. Considering that each harmful pathological response after SCI will hinder the recov-   writingoriginal draft (lead); writingreview and editing (lead).