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Article

Validation of a Precise Impactor in a Rodent Cervical Spinal Cord Injury Hemi-Contusion Model

by
Jose A. Castillo, Jr.
*,
Michael Nhien Le
,
Christopher Pivetti
,
Jordan E. Jackson
,
Edwin Kulubya
,
Zachary Paxton
,
Camille Reed
,
Khadija Soufi
,
Arash Ghaffari-Rafi
,
Allan Martin
,
Richard Price
,
Kee Kim
,
Diana Farmer
,
Aijun Wang
and
Rachel Russo
UC Davis Medical Center, Sacramento, CA 95817, USA
*
Author to whom correspondence should be addressed.
BioMed 2024, 4(4), 524-540; https://doi.org/10.3390/biomed4040039
Submission received: 24 October 2024 / Revised: 15 November 2024 / Accepted: 20 November 2024 / Published: 25 November 2024
Browse Figures
Versions Notes

Abstract

:
Background: Cervical spinal cord injuries (SCIs) are the most common type of human SCI. Although various animal SCI contusion models have been developed to mirror human pathology, few have described cervical-level injuries. This study aims to validate and establish optimal impact parameters to produce consistent incomplete cervical SCIs for testing novel therapies. Methods: Using a precise impactor, 3 cervical spinal cord hemi-contusions of varying severities were induced by modifying penetration depths and controlling dwell times. Penetration depths of 2.11 mm (n = 4), 2.24 mm (n = 4), and 2.36 mm (n = 3) were used with a dwell time of 0.05 s to create mild, moderate, and severe injuries. Behavioral assessments in weeks 1, 2, 5, and 8 included grooming test, forelimb asymmetry test, and the Irvine, Beatties, and Bresnahan forelimb scale (IBB). After 8 weeks, rats were euthanized, and spinal cord histology was performed. Results: Within each group, animals exhibited consistent motor deficits and functional recovery. Mean IBB scores varied significantly between each group at week 8 (p < 0.0001). Ipsilateral forelimb usage significantly improved throughout the study period in the mild (2.11 mm) and moderate (2.24 mm) groups, while the severely (2.36 mm) injured group continued to exhibit 100% asymmetrical forelimb usage. Conclusions: This study demonstrates that a precise impactor can create reproducible models of incomplete cervical SCIs. A penetration depth of 2.24 mm resulted in moderate injury with significant motor deficits that slowly improved over time, permitting future therapeutic studies in functional recovery.

1. Introduction

Spinal cord injury (SCI) is associated with severe disability and tremendous costs worldwide [1]. In the United States, the average cost of care during the first year for patients with SCIs ranges from $111,780 to $1,156,410 [2]. Spinal cord contusion is the most common human SCI, differing significantly from other mechanical injuries, such as compression, distraction, dislocation, or transection, that are employed in many animal studies [3]. Various SCI animal models in rodents are widely used for understanding pathophysiology and developing therapeutics [4,5,6,7]. However, most are thoracic SCI models [8]. In contrast, traumatic human SCI occurs most frequently at the cervical spine level, with 55% of cases affecting this region [9,10]. To effectively translate research findings to humans, it is important to develop animal models that accurately replicate the complexities of cervical SCI.
The primary aim of SCI research is to develop therapies that reduce motor deficits and enhance neurological recovery, while minimizing the number of animals used. An ideal SCI model should induce motor impairments that persist long enough to assess treatment effects but are still capable of showing measurable improvements over time [11].
Additionally, the severity of the injury should be moderate enough to avoid the need for early euthanasia for humane endpoints [12]. Furthermore, it is crucial for SCI models to produce consistent lesion characteristics with minimal variation within study groups, allowing reliable correlations between lesion severity and functional outcomes [11].
Contusion SCI models commonly utilize weight-drop or an impactor to transiently displace and damage the spinal cord [13,14,15]. This mimics the pathophysiological, functional, and morphological features of human SCI, including the absence of spontaneous neural regeneration and cystic cavity formation in the center of the spinal cord [16,17]. Current rodent contusion SCI models still have limitations which introduce undesirable variation in SCI lesions and functional outcomes. Weight drop models, for example, suffer from a rebound impact that leads to inconsistent injury severity and high within-group variability in outcomes [18,19]. Additionally, the lack of control over the impact duration and dwell time in central nervous system trauma limits the ability to fine-tune the severity of the injury, producing variability in both lesion characteristics and functional outcomes as well [20,21]. Improvements in these contusion models are essential for enhancing the reliability and translatability of SCI models, ultimately leading to more accurate assessments of novel therapeutic interventions.
Here, we validated a rodent cervical SCI hemi-contusion model, using a computer-controlled impactor (RWD Precise Impactor, Scintica Inc., Webster, TX, USA), with adjustable impact speed, depth, and dwell time to achieve reliable and accurate injuries. By testing three different penetration depths, we also aimed to determine an optimal penetration depth for producing a consistent and reliable injury of moderate severity to test therapeutic modalities.

2. Materials and Methods

2.1. Subjects

All experiments adhered to the National Institutes of Health Guide for the Care and Use of Animals and were approved by the UC Davis Institutional Animal Care and Use Committee. A total of 14 female Sprague Dawley® rats (200–250 g; Envigo, Indianapolis, IN, USA) were used in this study.
Rats were utilized instead of mice for modeling human SCI because the morphological and functional changes they exhibit following SCI more closely reflect those observed in humans. Unlike the mouse SCI model, where cells proliferate at the injury site to maintain contact between the transected spinal cord ends and prevent fluid-filled cyst formation [22,23], such responses are not seen in rats and humans, where cyst formation cranial and caudal to the injury site is common [24,25,26,27]. Furthermore, some studies suggest a limited degree of regeneration after a complete spinal cord transection in mice [28]. However, in rats and humans, complete severance of axonal connections results in permanent loss of motor and sensory function [29].
To minimize potential confounding factors related to weight and size discrepancies within the cohort, only female Sprague Dawley rats were selected for our study. This helps ensure consistency in spinal cord morphology, injury severity, and functional recovery post-injury. Matching male and female rats by weight would result in including developmentally younger males and older females, which could skew data outcomes. Notably, a previous study by Walker et al. demonstrated no significant differences in histological and functional outcomes between age-matched male and female Sprague Dawley rats in thoracic contusion SCI models [30]. Additionally, the use of male rats introduces potential challenges such as aggression, complex housing needs, and complications associated with bladder expression in most SCI models. Lastly, since our study does not involve interventions targeting sex hormones or receptors, female Sprague Dawley rats are preferable to maintain experimental consistency and minimize logistical challenges.

2.2. Surgical Procedure

Rats were induced and maintained on 2–5% isoflurane gas anesthesia and administered buprenorphine (0.05 mg/kg, s.c.) for pain regulation prior to surgical manipulation. Surgical sites were shaved and prepped with iodine and 70% ethanol. A 2–3 cm dorsal midline skin incision was performed, and the trapezius muscle was transected immediately lateral to the midline, from level C1 to T2. Spinous processes from C3 to T1 were exposed by muscular tissue blunt dissection. The spinal cord was exposed by C5 right dorsal hemi-laminectomy. A stereotactic frame (Scintica Inc., Webster, TX, USA) was used to stabilize the vertebral column while on the RWD Precise Impactor table. The impactor was fitted with a 2 mm diameter impactor rod tip, which was aligned over the exposed spinal cord. Penetration depths of either 2.11 mm, 2.24 mm, or 2.36 mm were programmed into the impactor’s computerized controller. These specific penetration depths were chosen because they induced varying degrees of motor impairment in the rats’ forelimb movement, with each depth producing distinct levels of functional response. Velocity and dwell time were determined based on penetration depth. The impactor pin was then fired at the prespecified parameters to create a C5 unilateral hemi-contusion injury. During impact, the rat was observed for forelimb flinching on the side of the injury, indicating successful contact between the impactor tip and the spinal cord. After impact, the rat was removed from the stereotactic frame, photographed under the microscope (Figure 1), and evaluated for bruising, subdural bleeding, or hematoma. The musculature layers and skin at the incision site were closed in a two-layer, running fashion with Vicryl® absorbable sutures (Ethicon, Somerville, NJ, USA). Surgical procedures were performed on a homeothermic blanket system (Kent Scientific, Torrington, CT, USA) to maintain 37 °C body temperature. Immediately after surgery, rats received 3 mL normal saline subcutaneously for fluid resuscitation.
After anesthesia recovery, animals were observed for right-sided forelimb paralysis, clubfoot, and forelimb orientation, then given a subsequent dose of buprenorphine for pain control, repeated twice daily for 2 days. Post-operatively, animals were inspected daily for 1 week, and then bi-weekly, for wound healing, weight loss, dehydration, autophagia, and discomfort, with appropriate veterinary care as needed. Rats were housed, with free access to food and water, in the Teaching Research and Animal Care Services vivarium at the UC Davis Medical Center. At the study endpoint, 8 weeks after surgery, euthanasia was performed by exsanguination under isoflurane anesthesia.

2.3. Motor Function Testing

2.3.1. General Testing Protocol

All motor function testing was performed and video-recorded with individual animals placed in a clear, circular enclosure (20 cm diameter by 40 cm wall height), with a mirror behind the cylinder angled to record movements from all directions. All tests were initially performed at least 1 week prior to surgical intervention to establish baseline values for comparison, and again after surgery on day 1 or 2 and weeks 1, 2, 5, and 8. Video recordings were blindly evaluated, and mean test scores were compared between groups at each time point, for each test, as follows.

2.3.2. Gross Motor Skills

The Irvine, Beatties, and Bresnahan (IBB) Forelimb Recovery (9-point) Scale was used to assess proximal and distal forelimb function, which is based on proximal forelimb movement, elbow position, and fine digit manipulation [31,32]. Briefly, rats were evaluated based on their ability to pick up and manipulate torus-shaped cereal (Cheerios™) and sphere-shaped cereal (Kix™) with their forelimb and digits. Subcategories of the IBB score were examined in further detail, and each was scored as either 0, 1, or 2 points, based on the individual category. Proximal forelimb function was assessed as extensive (2 points), slight (1 point), or no forelimb movement (0 points). Distal forelimb function was determined from grasping as normal (2 points), sometimes normal (1 point), or abnormal (0 points). Forepaw position was as follows: partially extended and adaptable (2 points), extended and non-adaptable (1 point), or clubbed (0 points).

2.3.3. Asymmetry Testing

Forelimb preference asymmetry was determined by evaluating the number of times the rat reared up to touch the wall with a forelimb in a span of 45 min: either ipsilateral or contralateral to the injury, or both [33], and expressed as the fraction of independent contralateral contacts out of total contacts.

2.3.4. Grooming Testing

A grooming test was used to evaluate overall mobility of the ipsilateral injury side forelimb [34], graded 0 to 5 based on the furthest point reached during self-contact, as follows: 0: no facial contact; 1: chin, but not mouth; 2: nose, beneath the eyes; 3: forehead, at or above the eyes; 4: in the front of ears; 5: behind the ears.

2.4. Tissue Processing

Immediately upon euthanasia at 8 weeks, transaortic perfusion (40 mL/min) was performed with phosphate buffered saline (PBS; 2 min), followed by 10% neutral-buffered formalin (NBF; 3 min). The spinal cord tissue was excised, post-fixed in 10% NBF, and cryoprotected in 30% sucrose PBS solution at 4 °C for 4–5 days. The injured segments of the spinal cord were transected along the axial plane through the center of the contusion. The proximal and distal segments were embedded side-by-side in a 2 cm cryomold with Tissue-Plus™ Optimal Cutting Temperature (O.C.T.) Compound (Fisher Healthcare, Houston, TX, USA). These blocks were stored at −80 °C, then sectioned into 20 µm axial slices on a cryostat (Leica Biosystems, Buffalo Grove, IL, USA) and mounted onto slides such that each slice contained a section of proximal cord and a section of distal cord. Every 5th, 10th, and 15th section of proximal and distal cord was retained for analysis. Sections were stained with Cresyl Violet Acetate Working Solution (Electron Microscopy Sciences, Hatfield, PA, USA) and mounted with Fisher Chemical™ Permount™ Mounting Medium (Fisher Healthcare, Houston, TX, USA).

2.5. Spinal Cord Histopathology

Light microscopy images of stained tissues were acquired at 5× magnification and used to assess SCI lesions using ImageJ software version 1.52p (National Institutes of Health, Bethesda, MD, USA). A blinded reviewer defined relevant anatomy for each segment of spinal cord examined, establishing the injured side as “ipsilateral” and the uninjured side as “contralateral” along a median plane. The reviewer then quantified the SCI lesion by outlining the hemi-contusion (inclusive of primary and secondary injury) within the ipsilateral half of each section of spinal cord and mathematically computed the lesion area. The area of intact spinal cord within the ipsilateral segment was determined by subtracting the injured area from the total area of the ipsilateral segment. The area of grey matter remaining within the intact ipsilateral cord was measured. The area of white matter was then calculated by subtracting the grey matter area from the intact ipsilateral spinal cord area. Quantitative cross-sectional area measurements of the intact spinal cord (grey and white matter) and lesion areas were performed for each of the retained sections and expressed as a mean. Motor neuron counts were determined for each of the retained tissue sections, including all cells with a diameter of >30 microns, on both the ipsilateral (injury) and contralateral sides.

2.6. Statistical Analysis

Statistical analysis and graphs created were performed using GraphPad Prism version 8 (La Jolla, CA, USA). Continuous outcomes were compared using a two-tailed t-test for parametric data, or a Kruskal–Wallis test for non-parametric data. Nominal data were assessed using Pearson’s chi-test or Fisher’s exact test if any expected cell count was less than 5. Multiple factors repeated ANOVA with Tukey–Kramer tests were used for comparison of the weekly IBB scores, asymmetry scores, and grooming scores. Independent linear correlation analyses between motor function outcomes and specific histologic outcomes were completed using Pearson correlation coefficients, with significance set at p < 0.05.

3. Results

3.1. Overall Cohort

Eleven rats underwent spinal cord hemi-contusion injury using the RWD Precise Impactor, with penetration depths of 2.11 (mild, n = 4), 2.24 (moderate, n = 4), and 2.36 mm (severe, n = 3) (0.4–0.5 m/s velocity; 0.05 s dwell time) (Table 1); 3 sham animals, rats that underwent hemi-laminectomy but not SCI, served as controls.

3.2. General Animal Health

Post-procedurally, all animals who received an injury immediately displayed functional deficits in the ipsilateral forelimb and paw. No respiratory deficits were observed; however, post-surgery stress responses included porphyrin deposits around the eyes and nose, which typically resolved within 5 days post-injury. All animals survived to the study endpoint.

3.3. Cohorts by Injury Severity

Of animals with mild SCI, 3 of 4 (75.0%) had visible bruising, and the remaining rats presented with subdural hematoma but no associated bruising (25.0%). The mean total anesthesia time was 41.8 min (SD 6.1). After anesthesia recovery, all 4 (100%) had right forelimb paralysis, right forelimb clubfoot, and a forward-facing right forelimb (Table 1).
Three of 4 moderately injured animals displayed bruising (75.0%); none showed evidence of subdural bleeding or subdural hematoma. The mean total anesthesia time was 39.3 min (SD 4.1). All 4 (100%) had right forelimb paralysis, right forelimb clubfoot, and a forward-facing right forelimb after anesthesia recovery (Table 1).
For the severe injury group, all 3 (100%) presented with bruising; none showed subdural bleeding or subdural hematoma. The mean total anesthesia time was 39 min (SD 3). All 3 (100%) had right forelimb paralysis, right forelimb clubfoot, and a forward-facing right forelimb after anesthesia recovery (Table 1).

3.4. Motor Function Test Results

After injury, fine forelimb and digit motor function were impaired in all injury groups.

3.4.1. Gross Motor Skills

IBB scores were significantly reduced in all SCI groups compared to the uninjured sham controls (Figure 2A). Across the 8-week study period, all injured groups showed progressively improving IBB scores; however, all scores remained significantly lower than the control group.
At week 1, mean IBB scores were similar in all injury groups (p = 0.42, Figure 2B). At week 2, significant differences appeared between the mild and the moderate groups (4.33 vs. 1.67; p = 0.02). At week 5, mean IBB scores in the mild group were significantly higher than both the moderate (5.75 vs. 3; p = 0.0027) and the severe groups (5.75 vs. 2.33; p = 0.0005), with no significant difference between the moderate and the severe groups (3 vs. 2.33; p = 0.8328). By week 8, mean IBB scores differed significantly in all injury groups: 6.75 vs. 4.5 (mild vs. moderate; p < 0.05); 6.75 vs. 2.33 (mild vs. severe; p < 0.0001); and 4.5 vs. 2.33 (moderate vs. severe; p < 0.05).

3.4.2. Fine Motor Skills

On assessment of finer forelimb movements, most animals displayed extensive forelimb movement with a subcategory IBB score of 2 points, with only 1 rat in the moderate group demonstrating slight movement (1 point) in week 1. No significant differences were found between groups (one-way ANOVA p = 0.6606) (Figure 3A,B).
We further assessed distal forelimb function, evaluated as forepaw position and grasping. In the first 2 weeks, forepaw position in all injury groups was significantly more abnormal compared to the control group (Figure 3C,D), with no significant differences between injury groups. In weeks 5 and 8, the 2.11mm group had normal forepaw position, which was similar to the normal controls. At week 5, both the moderate group and the severe group were significantly more abnormal compared to the mild group (p = 0.0160, p = 0.0011, respectively). At week 8, only a significant difference between the severe and the mild group remained (p = 0.0001). All animals in the severe group remained clubbed throughout the entire study period, which was significantly different from the normal controls (p = 0.0001, week 8).
Grasping also was significantly impaired in the injury groups. At weeks 1, 2 and 5, all groups were significantly more abnormal compared to controls. At week 8, the moderate and then severe groups remained significantly impaired (p = 0.0001, p < 0.0001, respectively). When compared to each other, there was a significant difference at week 8, with the severe groups being persistently abnormal, while the mild group was approaching always normal (p = 0.0036, Figure 3E,F).

3.4.3. Asymmetry Testing

When evaluating limb preference with the asymmetry test for the injured rats compared to the normal controls, at week 1 the percentage of times the rat used the contralateral forelimb increased from 19.3% in the control group to 98% in the severe (p = 0.0004), 83.3% in the moderate group (p = 0.0024), and 96% in the mild SCI group (p = 0.0031; one-way ANOVA p = 0.0400, Figure 4A). This significant increase in asymmetry percentage remained in the severe group throughout the entire study period. The moderate group was significantly more asymmetrical at weeks 1 and 2 only when compared to normal controls. And the mild group was only significantly more asymmetrical at week 1, but then at weeks 2–8 was not significantly different from the normal controls. Conversely, the severe group remained at around a 100% asymmetry percentage without any improvement over time. When comparing injury groups, there was only one comparison for asymmetry data that was significantly different, which was comparing the mild group to the severe group at week 5 (46% vs. 99%, p = 0.0409, Figure 4B).

3.4.4. Grooming Testing

With regards to grooming, both the moderate and the severe groups had significantly reduced grooming scores when compared to the normal controls throughout the entire study period (Figure 5A). The mild group was similar to the control group in its grooming scores, as from week 1 all the way through week 8 there were no significant differences between the two groups. There were no significant differences when comparing all groups to one another at week 1; however, in week 2, the mild group had a significantly higher grooming score when compared to both the moderate (4.75 vs. 2.5, p = 0.0091) and severe groups (4.75 vs. 1.67, p = 0.0010), which remained significant at week 5. At week 8, only mild compared to severe was significantly different (4.25 vs. 2, p = 0.0164).

3.5. Spinal Cord Histopathology Results

3.5.1. Lesion Void and Remaining Intact Spinal Cord

All injured groups had significantly more spinal cord tissue void compared to normal controls (one-way ANOVA p = 0.023); however, there were no significant differences when comparing each group to one another (Figure 6A). Similarly, even though there were no significant differences in overall void area between injured groups, they did have a higher total void area compared to normal controls (one-way ANOVA p = 0.0265, Figure 6B).
With regards to the remaining intact spinal cord, upon quantification all injury groups had significantly less gray matter compared to normal controls, without any significant difference between groups (Figure 7A). Interestingly, only the moderate and the severe penetration groups had a significantly lower amount of white matter remaining when compared to the normal controls (Figure 7B). However, when evaluating the overall ipsilateral intact spinal cord, there was a significantly reduced amount in all injured groups compared to normal controls (Figure 7C).

3.5.2. Motor Neuron Counts

The number of motor neurons was significantly decreased on the ipsilateral injured side of all injured groups compared to normal controls (one-way ANOVA p = 0.0176); however, there were no differences in motor neuron counts between the injury groups (one-way ANOVA p = 0.7091). We compared the ipsilateral injury to the contralateral non-injured side within groups and found that each group had a significantly decreased number of motor neurons on the ipsilateral injured side. In the mild group, the contralateral side had an average of 11.83 motor neurons compared to 0.08 on the injured side (p = 0.0286). Similarly, there was decrease from 12.30 on the contralateral side to the 0.42 on the injured side for the moderate group (p = 0.0286). Lastly for the severe group, there was a drop from 13.75 motor neurons to none on the injured side (p = 0.0286). Figure 8 is an image of the respective motor neuron counting histology sections.

3.6. Correlation Between Histology and Motor Function

The Pearson correlation tests found significant correlation between motor function outcomes and the total intact ipsilateral spinal cord area (r2 = 0.70, p = 0.0002, Figure 9A). Additionally, functional outcomes showed significant correlations with both the remaining area of gray matter (r2 = 0.36, p = 0.02) and white matter (r2 = 0.77, p < 0.0001, Figure 9B,C).

4. Discussion

Our study evaluated the motor function and histopathological outcomes of three different penetration depths of cervical injury in a rat spinal cord hemi-contusion model with a computer-controlled impactor (RWD Precise Impactor). We found a consistent degree of motor impairment, extent of functional recovery, and speed of recovery within each group. There were significant differences in motor function between the groups that correlated with the depth of penetration. In addition to motor and behavioral deficits, we observed that the amount of remaining intact ipsilateral spinal cord, including both gray and white matter, was not only dependent on the injury severity but also correlated with functional motor outcomes. The moderate injury group (2.24 mm penetration depth) exhibited significant impairments, at times similar to the severe injury group, that improved slowly, making it the ideal characteristic for future interventional studies that test the effects of therapies aimed at reducing secondary SCI. Overall, our study validated that this technology and technique can reproduce injuries of varying severities, providing a reliable model for future therapeutic studies in SCI.

4.1. A Highly Reproducible Injury

Existing rodent contusion SCI models have weaknesses that limit their utility and reproducibility. Contusion SCI models typically utilize weight-drop or an impactor to transiently displace and damage the spinal cord [13,14]. The weight-drop model is often plagued by a variable secondary rebound and additional impact that induces undesirable variation in the SCI lesions, as seen in the Multicenter Animal Spinal Cord Injury Study (MASCIS) [18,19]. Subsequent iterations have improved the weight-drop design; however, the impact duration still cannot be precisely controlled. Force-controlled impact models do not experience a bounce artifact but require starting with a “dural touchpoint” that imparts undesirable variation during impact [35,36,37]. The computer-controlled hemi-contusion spinal cord impact model ideally addressed several of these challenges. The stereotactic frame established a reproducible starting point and stabilized the vertebral column, while the computerized impact pin controller delivered reproducible impact parameters with precise depth, dwell time, and velocity that we could vary as desired. The within-group variation was also small when analyzing functional outcomes in this study. This was evident by the fact of a consistent degree of motor impairment, extent of functional recovery, and speed of recovery within each group
The computer-controlled impactor also offered an advantage through the precise control over the impact speed, depth, and dwell time. We found our parameters produced consistent and graded cervical hemi-contusions in our rat model. The precise control of the impactor was particularly important for evaluating the injury immediately post-impact. Previous studies suggest that subdural hematomas (SDH) can exacerbate SCI outcomes [38]. In our study, only 1 out of 11 rodents developed an SDH. Additionally, we also found a similar void area for each rat in their respective groups. This finding demonstrates that the technique is reproducible for cervical SCI of varying severities.

4.2. Depth of Penetration Corresponds to Severity of Impairments

We were able to achieve three different severities of motor deficits across three separate motor function tests with increasingly severe deficits positively correlating with increasing impact depth. Other groups employing cervical spinal cord contusions with a different impactor (Ohio State University Electromagnetic Spinal Cord Injury Device) have found a similar pattern of more severe injury corresponding to worsening motor score using functional assessments such as the IBB [39]. In thoracic contusion models, motor function scores were also consistently graded, with mild groups being significantly greater than for moderate injury groups throughout the entire study period [35,40]. In each of these studies, increasing penetration depth exhibited a dose-response relationship in IBB scores, suggesting that motor outcomes were dependent on injury penetration depth, though alterations in velocity and dwell time have been less studied [37].
Using our defined parameters, we found that animals with mild injury (2.11 mm penetration depth) rarely exhibited significant difference in motor scores compared to the control animals. This model may be well-suited to the study of SCI exacerbating factors, such as ischemia-reperfusion injury or hemorrhagic shock, on motor outcomes. The moderate injury group (2.24 mm penetration depth) exhibited significant impairments that improved slowly with time, achieving ideal characteristics for future interventional studies that test the effects of therapies aimed at reducing secondary SCI. Animals with severe injury (2.36 mm deep penetration) consistently exhibited persistent dense deficits. Such a model could be desirable for testing interventions, such as exoskeletons or extremity re-animation, that require a stable, severe injury. Furthermore, by employing a unilateral or hemi-contusion injury we could complete a detailed analysis of motor impairments, comparing the injured limb to the uninjured limb as an internal control, while minimizing the overall severity of injury.

4.3. Functional Deficits Correspond to Histological and Anatomical Injury Severity

Across our three groups, we found the degree of functional deficits positively correlated with the extent of histological and anatomical injury. While this finding is not surprising, correlating functional observations with histological and anatomical findings is important validation for our model. In our hemi-contusion model, we could compare tissues within the same individual at the same level of the spinal cord, with the uninjured hemi-cord serving as the internal control to better understand structural drivers of function. We found no difference between injury groups with regards to the lesion void area or motor neuron counts within the ipsilateral cord sections, though neuronal loss was significantly greater on the side of injury compared to the contralateral uninjured hemi-cord. Furthermore, a decreasing area of remaining intact ipsilateral spinal cord, white matter, and gray matter, correlated with more severe motor deficits, similar to other cervical contusion models [35,41]. This data supports the fact that it is the area of remaining intact ipsilateral cord area (white matter area and gray matter area) rather than the loss of motor neurons that are driving the behavioral and motor function outcomes.

4.4. Limitations

Several limitations should be considered in our study. The sample size for each injury group was relatively small, which limits the statistical power and generalizability of the findings. For our sample size, though small, one of the benefits is that the intragroup variation is small so the sample size can ideally be low. This also is relevant because it upholds the reduction principle for ethical animal use. Ultimately, we were able to have meaningful statistical comparisons with just a few animals, but readers should note this situation when drawing a conclusion, and reproductions of this project should weigh the power of using a larger population. Secondly, the study only assessed three different impact penetration depths. While these depths were sufficient to produce mild, moderate, and severe injuries, it is possible that other depths could produce more nuanced injury severities that may be more relevant for very specific therapeutic investigations. Future studies could increase the cohort size, explore a wider range of injury severities, and perform direct comparisons of our protocol to pre-existing models to better understand the spectrum of spinal cord damage and recovery outcomes.

5. Conclusions

Our study demonstrated the ability to create a range of graded injury severities with different penetration depths using a computer-controlled impactor (RWD Precise Impactor), each with potential applications as a pre-clinical model. The injuries were reproducible and resulted in significant and quantifiable differences in motor function, anatomical, and histological features of injury severity correlating with increasing impact depth. For our purposes, a 2.24 mm penetration depth resulted in a moderate injury with the optimal characteristics to permit evaluation of the therapeutic efficacy of novel SCI treatments.

Author Contributions

Conceptualization, writing, major revisions, data acquiring/re-acquiring and analysis/re-analysis—J.A.C.J., M.N.L. and C.P.; Conceptualization, writing, data acquiring and analysis—J.E.J.; Writing—review and editing—E.K., Z.P., C.R., K.S., A.G.-R., A.M., R.P., K.K., D.F., A.W. and R.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of University of California, Davis (protocol code 23028 and approved on 3 October 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Gross injury of the cervical spinal cord contusion on a rat caused by the precise impactor.
Figure 1. Gross injury of the cervical spinal cord contusion on a rat caused by the precise impactor.
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Figure 2. Irvine, Beatties, and Bresnahan (IBB) scores. Scores all measured on a scale of 0–9, and within each group the mean score is plotted by week. Preop indicated pre-injury time-point. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) All groups are compared to the normal controls. (B) All groups (2.11 mm, 2.24 mm, and 2.36 mm) are compared to one another.
Figure 2. Irvine, Beatties, and Bresnahan (IBB) scores. Scores all measured on a scale of 0–9, and within each group the mean score is plotted by week. Preop indicated pre-injury time-point. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) All groups are compared to the normal controls. (B) All groups (2.11 mm, 2.24 mm, and 2.36 mm) are compared to one another.
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Figure 3. Specific Irvine, Beatties, and Bresnahan (IBB) scores. Mean scores across all animals within 1 group per week were plotted. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) Forelimb movement with all groups compared to normal controls. (B) Forelimb movement with all groups (2.11 mm, 2.24 mm, and 2.36 mm) compared to one another. (C) Forepaw movement with all groups compared to normal controls. (D) Forepaw movement with all groups (2.11 mm, 2.24 mm, and 2.36 mm) compared to one another. (E) Grasping position with all groups compared to normal controls. (F) Grasping position with all groups (2.11 mm, 2.24 mm, and 2.36 mm) compared to one another.
Figure 3. Specific Irvine, Beatties, and Bresnahan (IBB) scores. Mean scores across all animals within 1 group per week were plotted. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) Forelimb movement with all groups compared to normal controls. (B) Forelimb movement with all groups (2.11 mm, 2.24 mm, and 2.36 mm) compared to one another. (C) Forepaw movement with all groups compared to normal controls. (D) Forepaw movement with all groups (2.11 mm, 2.24 mm, and 2.36 mm) compared to one another. (E) Grasping position with all groups compared to normal controls. (F) Grasping position with all groups (2.11 mm, 2.24 mm, and 2.36 mm) compared to one another.
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Figure 4. Forelimb asymmetry test. Percent calculated by contralateral contacts divided by the total number of contacts. Mean asymmetry percentages across all animals within one group per week were plotted. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) All groups are compared to the normal controls. (B) All groups (2.11 mm, 2.24 mm, and 2.36 mm) are compared to one another.
Figure 4. Forelimb asymmetry test. Percent calculated by contralateral contacts divided by the total number of contacts. Mean asymmetry percentages across all animals within one group per week were plotted. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) All groups are compared to the normal controls. (B) All groups (2.11 mm, 2.24 mm, and 2.36 mm) are compared to one another.
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Figure 5. Grooming test. Mean grooming score across all animals within one group per week were plotted. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) All groups are compared to the normal controls. (B) All groups (2.11 mm, 2.24 mm, and 2.36 mm) are compared to one another.
Figure 5. Grooming test. Mean grooming score across all animals within one group per week were plotted. *, #, ** denotes p < 0.05 for group comparisons using repeated measure analysis of variance and t-tests. (A) All groups are compared to the normal controls. (B) All groups (2.11 mm, 2.24 mm, and 2.36 mm) are compared to one another.
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Figure 6. Histological analysis of the void area after injury. (A) Main void area representation by group (* p < 0.0001; ** p < 0.0001; *** p < 0.0001). All groups had significantly higher main void area compared to normal controls (one-way ANOVA p = 0.023). (B) Total void area representation by group, which included any other lesions or microcysts. All groups had significantly higher total void area compared to normal controls (one-way ANOVA p = 0.0265).
Figure 6. Histological analysis of the void area after injury. (A) Main void area representation by group (* p < 0.0001; ** p < 0.0001; *** p < 0.0001). All groups had significantly higher main void area compared to normal controls (one-way ANOVA p = 0.023). (B) Total void area representation by group, which included any other lesions or microcysts. All groups had significantly higher total void area compared to normal controls (one-way ANOVA p = 0.0265).
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Figure 7. Histological analysis of remaining intact spinal cord. (A) Gray matter area with all injury groups having significantly lower remaining ipsilateral gray matter compared to normal controls (* p < 0.0001; ** p < 0.0001; *** p < 0.0001). (B) White matter area with only the 2.24 mm (** p = 0.0189) and the 2.36 mm (*** p = 0.0142) having significantly less white matter. (C) Total ipsilateral remaining intact spinal cord was significantly lower for all treatment groups when compared to normal controls (* p = 0.0045; ** p = 0.0006; *** p = 0.0008).
Figure 7. Histological analysis of remaining intact spinal cord. (A) Gray matter area with all injury groups having significantly lower remaining ipsilateral gray matter compared to normal controls (* p < 0.0001; ** p < 0.0001; *** p < 0.0001). (B) White matter area with only the 2.24 mm (** p = 0.0189) and the 2.36 mm (*** p = 0.0142) having significantly less white matter. (C) Total ipsilateral remaining intact spinal cord was significantly lower for all treatment groups when compared to normal controls (* p = 0.0045; ** p = 0.0006; *** p = 0.0008).
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Figure 8. Cresyl violet stains of injured spinal cord for each respective depth. (A) Normal, (B) 2.11 mm, (C) 2.24 mm, (D) 2.36 mm. Panel E is zoomed in on the black box in panel (A). Scale bar for panels (A–D) = 2 mm and for panel (E) = 100 um. Black arrows in panel (E) show representative motor neurons that are 30–70 um in diameter.
Figure 8. Cresyl violet stains of injured spinal cord for each respective depth. (A) Normal, (B) 2.11 mm, (C) 2.24 mm, (D) 2.36 mm. Panel E is zoomed in on the black box in panel (A). Scale bar for panels (A–D) = 2 mm and for panel (E) = 100 um. Black arrows in panel (E) show representative motor neurons that are 30–70 um in diameter.
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Figure 9. Linear correlation analyses between IBB score and specific histology parameters. Movement was assessed according to the IBB score: no movement (0 points), slight movement (1 point), extensive movement (2 points). (A) Remaining intact ipsilateral cord area was positively correlated with IBB score. (B,C) Gray and white matter were also positively correlated with IBB score.
Figure 9. Linear correlation analyses between IBB score and specific histology parameters. Movement was assessed according to the IBB score: no movement (0 points), slight movement (1 point), extensive movement (2 points). (A) Remaining intact ipsilateral cord area was positively correlated with IBB score. (B,C) Gray and white matter were also positively correlated with IBB score.
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Table 1. Injury characteristics by rat.
Table 1. Injury characteristics by rat.
ID #Total Anesthesia Time (Min)Impact Penetration Depth (mm)Impact Velocity (m/s)Dwell Time (s)Bruise (Y/N)Subdural Bleeding (Y/N)Subdural Hematoma (Y/N)R. Forelimb Paralysis (Y/N)Clubfoot (Y/N)Forward Or BackwardFlinch (Y/N)
1462.110.40.05YYNYYForwardY
2482.110.50.05YNNYYForwardY
3362.110.50.05NNYYYForwardY
4372.110.50.05YNNYYForwardY
5392.240.50.05YNNYYForwardY
6442.240.50.05NNNYYForwardY
7342.240.50.05YNNYYForwardY
8402.240.40.05YNNYYForwardY
9362.360.40.05YNNYYForwardY
10422.360.40.05YNNYYForwardY
11392.360.40.05YNNYYForwardY
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Castillo, J.A., Jr.; Le, M.N.; Pivetti, C.; Jackson, J.E.; Kulubya, E.; Paxton, Z.; Reed, C.; Soufi, K.; Ghaffari-Rafi, A.; Martin, A.; et al. Validation of a Precise Impactor in a Rodent Cervical Spinal Cord Injury Hemi-Contusion Model. BioMed 2024, 4, 524-540. https://doi.org/10.3390/biomed4040039

AMA Style

Castillo JA Jr., Le MN, Pivetti C, Jackson JE, Kulubya E, Paxton Z, Reed C, Soufi K, Ghaffari-Rafi A, Martin A, et al. Validation of a Precise Impactor in a Rodent Cervical Spinal Cord Injury Hemi-Contusion Model. BioMed. 2024; 4(4):524-540. https://doi.org/10.3390/biomed4040039

Chicago/Turabian Style

Castillo, Jose A., Jr., Michael Nhien Le, Christopher Pivetti, Jordan E. Jackson, Edwin Kulubya, Zachary Paxton, Camille Reed, Khadija Soufi, Arash Ghaffari-Rafi, Allan Martin, and et al. 2024. "Validation of a Precise Impactor in a Rodent Cervical Spinal Cord Injury Hemi-Contusion Model" BioMed 4, no. 4: 524-540. https://doi.org/10.3390/biomed4040039

APA Style

Castillo, J. A., Jr., Le, M. N., Pivetti, C., Jackson, J. E., Kulubya, E., Paxton, Z., Reed, C., Soufi, K., Ghaffari-Rafi, A., Martin, A., Price, R., Kim, K., Farmer, D., Wang, A., & Russo, R. (2024). Validation of a Precise Impactor in a Rodent Cervical Spinal Cord Injury Hemi-Contusion Model. BioMed, 4(4), 524-540. https://doi.org/10.3390/biomed4040039

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