**The Effect of Botulinum Neurotoxin Serotype a Heavy Chain on the Growth Related Proteins and Neurite Outgrowth after Spinal Cord Injury in Rats**

#### **Ya-Fang Wang, Fu Liu, Jing Lan, Juan Bai and Xia-Qing Li \***

Department of Pathophysiology, Shanxi Medical University, Taiyuan 030001, China; 041510570@b.sxmu.edu.cn (Y.-F.W.); 031513156@b.edu.cn (F.L.); 041410136@b.sxmu.edu.cn (J.L.); 041510569@sxmu.edu.cn (J.B.)

**\*** Correspondence: xqli2013@sxmu.edu.cn; Tel.: +86-135-4632-8095

Received: 23 November 2017; Accepted: 31 January 2018; Published: 2 February 2018

**Abstract:** (1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (*p* < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (*p* < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.

**Keywords:** botulinum neurotoxins; botulinum neurotoxin serotype A; heavy chain; botulinum neurotoxin serotype a heavy chain (BoNT/A HC); spinal cord injury (SCI); nerve regeneration; growth associated protein 43 (GAP-43); superior cervical ganglion 10 (SCG10); neuronal processes; neural regeneration

**Key Contribution:** This is a preliminary and breakthrough explore for the effect of botulinum neurotoxin serotype A heavy chain on neuritogenesiis in vivo. The local application of BoNT-A heavy chain to the site of spinal cord injury might be helpful for relief of structural damage and dysfunction.

#### **1. Introduction**

A spinal cord injury (SCI) is a devastating condition characterized by a sudden loss of spinal functions distal to the level of trauma, including sensory, motor, and autonomic function. These effects are due to various traumatic or non-traumatic factors. The regenerative capacity of the injured spinal cord is extremely limited in adult mammals [1,2]. Patients suffer various disabilities which greatly impact quality of life [3]. The deficiency of spinal regenerative ability after an SCI has been attributed to various alterations in the local microenvironment, including: (1) the accumulation of many inhibitory factors by the degeneration of neurons, axon, and myelin; and (2) the lack of a sufficient number of upregulated or activated neuritogenic-associated genes and proteins [4–8]. At present, although there are many attempts for the treatment of the SCI, such as IL-6, stem cell transplantation [9,10], etc. few are actually available for clinical practice. Therefore, endeavors towards new strategies to revitalize neuritogenic ability following spinal injuries are being continuously made.

Botulinum toxin A (BoNT/A) is one of seven botulinum toxins (categorized based on serologic features) and is one of the most potent toxins known [11–14]. The toxicity of BoNT/A is due to its selective binding to the membranous receptor located on the presynaptic membrane of the motor endplate. This leads to the cleavage of the synaptosomal-associated protein 25 kDa (or SNAP-25) which blocks the release of the neurotransmitter acetylcholine contained in the vesicles [15,16]. This results in the paralysis of skeletal muscle. Patients die of severe respiratory failure due to a lack of respiratory muscle contractions [17]. Besides the aforementioned mechanisms of BoNT/A, it has been verified that it can also inhibit or block the release of other components in synaptic vesicles of different neurons, such as the calcitonin gene related peptide (CGRP) and substance P in sensory neurons [18–22]. Because of the actions of BoNT/A on neurotransmitters, it has been used as therapy to relieve hyper-reactive muscle dysfunctions (e.g., torticollis and hemifacial spasm), various types of chronic pain, hyperhidrosis, etc. [23,24].

Structurally, BoNT/A consists of two polypeptide chains linked by a disulfide bond. The longer chain (MW = 100 kDa) is called a heavy chain (HC) of BoNT-A; it is mainly responsible for binding to target cells and mediating the endocytosis of the toxin. The other chain with a smaller molecular weight is a light chain (LC, MW = 50 kDa). The LC is a zinc-dependent proteolytic enzyme which contributes to the cleavage of SNAP-25 at the neuromuscular junction [15,25,26]. It had been observed by some investigators that new axonal branches formed around skeletal muscles that had been paralyzed by BoNT/A for three to four months [27–29]. Meanwhile, the observations in vitro showed that BoNT/A was able to promote neuritogenesis in mouse motor neuron cultures [30]. The observation of neurite sprouting associated with BoNT/A in vivo or in vitro could not simply be attributed to the toxic role of BoNT-A. Moreover, the observations form Coffield's lab also showed that the isolated binding domain heavy chain (BoNT/A HC) has the same role as BoNT/A [30]. The signal pathways relating to the binding of the heavy chain and the receptor might also be a reasonable alternative explanation.

In our previous studies, the increased phosphorylation of Akt and ERK1/2 (growth- and survival-related intracellular signal proteins) was paralleled with neuritogenesis when adding BoNT/A heavy chain into neuro-2a cell cultures (data published in Chinese). In that study, an obvious increase in phosphorylated-ERK 1/2 was seen between one to five hours after adding 1 nmol/L of BoNT/A HC (*p* < 0.05), and an increased protein level of phosphorylated-Akt was observed mainly at 15 and 60 min (*p* < 0.05). Based on the results, it was concluded that the BoNT-A heavy chain stimulates neuritogenesis. The neuritogenic mechanism of it on Neuro-2a cells might be due to the activation of ERK1/2 and Akt phosphorylation. Meanwhile, it is reasonably presumed that BoNT/A heavy chain exerted its biological roles by modulating many intracellular signals and proteins expression, some of which may be involved in neural growth and regeneration; others are possibly related to growth inhibition. Among of these proteins, growth-associated protein-43 (GAP-43) and superior cervical ganglion 10 (SCG 10) have already been evidenced to be closely related to trhe axonal growth and neural regeneration [31–33]. Therefore, commercial recombinant BoNT/A HC was applied in this study to explore the effect of BoNT/A HC on the expression of some selective growth-associated

#### *Toxins* **2018**, *10*, 66

proteins, as well as its role in neurite outgrowth in rat lumbar SCI model in vivo. Meanwhile, the motor and sensory functions of the ipsilateral hindlimb were evaluated after BoNT/A HC treatment as a therapeutic regent.

#### **2. Results**

#### *2.1. Unilateral Lumbar SCI Model and Spinal Function Evaluation*

Observations of the SCI two days after surgery showed that it was located within the left lumbar area (Figure 1A,B). The control values for the grasp power and thermal hyperalgesia were obtained one day prior to the SCI. Two days after the SCI, both motor and sensory function appeared remarkably impaired (Figure 1C,D), i.e., the value of grasp power on grip test appeared dropped, which indicated the dysfunction of nervous control on skeletal muscle in hindlimbs, which might be related to the lesion of the corticospinal tract at lumbar spinal level, and also contributed in the appearance of the draging of the ipsilateral hindlimb.

**Figure 1.** An observation of lumbar spinal cord injury (SCI) and spinal function testing. (**A**) The SCI was made by the tip of a pair of fine forceps. A clear unilateral defection was seen in the field (arrow explanation). (**B**) The lesion was located in the lumbar area when the whole spinal cord was exposed (both two arrows point to the same damaged area). (**C**,**D**) There was an obvious ipsilateral drop in the grip score and a prolonged hind-paw withdrawal latency greater than 25 s (# *p* < 0.05 versus the control group; bar = 5 mm).

#### *2.2. The Alteration of Local Protein Expression after SCI and the Effect of BoNT/A HC on It*

Two-dimensional electrophoresis with silver nitrate staining showed that the general expression of protein in SCIs was altered compared to that of the control group. The major changes noted after injury were that some protein dots were lighter in color or smaller in size, while others appeared simultaneously denser or increased. Furthermore, the proteins with either increased or decreased expression appeared as a single dot or a dot group. After administration of the BoNT/A HC, the alterative expression of proteins demonstrated some interesting phenomena: some of the protein dots with increased expression tended to decrease towards the levels seen in the control group, whereas some dots with increased expression either further increased remarkably or went down in expression. It was noted that the protein dots that were 35–45 kDa (isoelectric point of 4 to 5) and 18–25 kDa in molecular weight (isoelectric

point 7) had an interesting appearance: their expression was increased after the SCI, and then this increase became much more significant after treatment with BoNT/A HC (Figure 2).

**Figure 2.** The two-dimensional gel with silver nitrate staining. (**A**) The images show that the protein dots change to molecular weights of approximately 18–45 kDa. Red and green arrows point to the dots with obvious alterative expression in the different groups. (**B**) The integrated OD value for protein expression (*n* = 4/each group) appeared at approximately 18–25 kDa. (**C**) The integrated OD value for protein expression appeared at approximately 35–45 kDa. Results were statistically significant ( # *p* < 0.05, vs. control group; \* *p* < 0.05, vs. both control & injury groups).

#### *2.3. Effects of BoNT/A Heavy Chain on the Expression of Selective Growth-Associated Proteins*

According to the profile of the 2D gel from different group, two dots from the molecular weight (MW) between 35 and 45 kDa, Isoelectric point (pI) around 4–5 and the MW between 18 and 25 kDa, pI around 7 were selected as the aimed proteins. Based mainly on the molecular weight of some growth-associated proteins, GAP43 (MW = 43 kDa) and SCG10 (MW = 20.8 kDa) were selected as target proteins for the BoNT/A HC treatment intervention. Both SDS-PAGE and a Western blot showed that the expression of p-GAP43 and SCG10 were increased two days after the SCI while using GAPDH as a loading control. However, the increase declined as time post-injury increased, even dropping below the level of the control group two weeks after the injury. With the BoNT/A HC injection, there was a much greater increase of the expressions of the two proteins. The increase was significant compared with SCI-only samples during the corresponding period (*p* < 0.05). In comparison, SCG10 had a more increased expression than p-GAP43 after administration of BoNT-A HC; this was based on the semi-quantification of both protein bands on the Western blot. The expression level of SCG10 was almost two times greater than that of control groups and 75% greater than SCI-only groups at four weeks post-injury. Meanwhile, the expression of p-GAP43 was also increased after BoNT/A heavy chain treatment, but the change was not as dramatic as in SCG10 (Figure 3).

**Figure 3.** The effect of BoNT-A heavy chain (HC) on the expression of p-GAP43 and SCG10. The Western blot bands for both: p-GAP43 (**A**); and SCG10 (**B**) at different periods (two days, one week, two weeks, and four weeks). The semi-quantification analysis (4 sample/each group) of: p-GAP43 (**C**); and SCG10 (**D**). Results were statistically significant (# *p* < 0.05, vs. the control group; \* *p* < 0.05, vs. both the control and the injury groups).

#### *2.4. The Distribution of Positive GAP43 and SCG10 Immunofluorescence at the Injured Area after Administration of the BoNT/A HC*

Results showed that in a normal spinal cord both GAP43 and SCG10 are evident in only a few cells with weak positive expression. Under the microscope, the positive immunofluorescence of GAP43 and SCG10 at the injury area was consistent with the results of the Western blot. The distributive features of the two proteins appeared very similar. The positive GAP43 and SCG10 expression was mainly within the cell body. These cells were very evident during the immunofluorescence of both proteins; they were found in the caudal and rostral parts of the injury area two to seven days after the injury, with or without BoNT/A HC administration. With BoNT/A HC treatment, however, the immunopositivity of both proteins appeared more intense than the SCI-only groups after two weeks post-injury. Furthermore, with BoNT/A heavy chain treatment, the positive staining was observed not only within the cytoplasm but also in the cellular processes; this made the processes easily identifiable. Comparably, the neuronal processes were much more obvious in the immunofluorescence of SCG10 than of GAP43 (Figure 4). Both Western blot and immunofluorescence results imply that SCG10 might be an important growth-associated protein affected by the BoNT/A HC. The positive SCG10 neuronal processes were used as the target for the measurement of neurite length and number after in vivo administration of BoNT/A HC.

**Figure 4.** A comparison of the immunofluorescence of GAP43 (Green) and SCG10 (Red) at the spinal injury site. The scale bars were set at 500 μm (on the long rectangular image at right bottom; and 20 μm (short rectangular image at right bottom; lower sections are the magnified images from rostral and caudal sites of the corresponding upper one. Each group consists of one long rectangular and two short rectangular images), respectively.

#### *2.5. The Measurement of SCG 10 Positive Neuronal Processes*

Based on immunofluorescence, the length and number of neuronal processes with SCG10 positivity at the caudal and rostral areas of injury were imaged and measured using ImageJ software (a free software from https://imagej.nih.gov/ij/). (The number of SCG 10 positive neurons was various from group to group, but the variation between group and group, especially SCI and BoNT/A HC treatment group, was not significant (*p* > 0.05). The calculation appeared as 35–45 (in controls), 55–65 (in SCI 2 d), 80–90 (SCI 1 w), 90–100 (SCI 2 w), 80–90 (SCI 4 w), 70–80 (with BoNT/A HC 2 d), 100–105 (with BoNT/A HC 1 w), 100–105 (with BoNT/A HC 2 w) and 90–100 (with BoNT/A HC 4 w). SCG10 positive processes were scattered, short, and not easily identified one week after the SCI; this was true even regarding

the positive cells that were clearly observed in that time. With lasting post-injury time (four weeks), these neuronal processes eventually disappeared in the SCI-only group. In contrast, the number of neuronal processes significantly increased when BoNT/A HC was given. The total length of positive SCG10 neurites reached 400 μm in four weeks, and the process branches were observed to increase even two weeks after injury. The increase in neuronal number was almost three times greater than control and SCI-only groups (*p* < 0.05) (Figure 5). Additionally, the percentage of positive SCG10 cells with identified neurites was higher than in SCI-only groups at the corresponding time (*p* < 0.05) (Figure 5). These results indicate that BoNT/A HC was able to stimulate the re-growth of neuronal processes after an SCI, as previously seen in vitro studies.

**Figure 5.** The effects of BoNT/A HC on the neurite outgrowth. (**A**) The visualization of neuronal processes and their variation in different groups using SCG10 immunofluorescence. A quantitative comparison of the: total length (**B**); number (**C**); and the percentage of neurons with visualized processes in all SCG10 positive cells (**D**). Results were statistically significant (# *p* < 0.05, vs. control group; \* *p* < 0.05, vs. both control and injury groups); Bar = 20 μm.

#### *2.6. The Amelioration of Motor and Sensory Function by BoNT/A HC Treatment Based on SCI*

The ipsilateral hindlimb motor function was evaluated using grip testing at two days, one week, two weeks, and four weeks post-SCI. Results showed that there was a significant drop in grip scores, which indicated that there was some damaged motor function of the ipsilateral hindlimb after the SCI. However, rats administered with BoNT/A HC treatment showed relatively higher scores than those in the SCI-only group (*p* < 0.05), though their scores were not as high as those of the control group (Figure 6).

The test for thermal hyperalgesia was used to assess sensory function. The results indicated that the paw withdrawal latency (PWTL) was prolonged after an SCI, with or without the application of BoNT/A HC, and there was no significant difference between the SCI groups and the BoNT/A HC treatment groups (Table 1).

**Figure 6.** The motor function changes in the ipsilateral hindlimb after BoNT/A HC treatment. Results were statistically significant (# *p* < 0.05, vs. control group; \* *p* < 0.05, vs. injury group).


