Comparative Analysis of S100β Expression on Spinal Cord Tissue of Injured Vs. Uninjured Rat Models (Rattus Norvegicus Sprague-Dawley)

doi:10.21203/rs.3.rs-5281572/v1

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Comparative Analysis of S100β Expression on Spinal Cord Tissue of Injured Vs. Uninjured Rat Models (Rattus Norvegicus Sprague-Dawley)

https://doi.org/10.21203/rs.3.rs-5281572/v1

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Background: S100β is a Ca2+-binding protein found in glial astrocytes of the central nervous system. It is known to increase in various pathological conditions, including spinal cord injury (SCI), and is commonly detected in cerebrospinal fluid and serum. However, no study has examined histopathological S100β expression in spinal cord tissue after SCI.

Objective:This study aimed to assess differences in S100β expression in spinal cord tissue of rat (Rattus norvegicus Sprague-Dawley) SCI models compared to uninjured controls.

Method: An experimental post-test only randomized control group design was used. Thirty Sprague-Dawley rats were divided into three groups: control (K), SCI induction with termination at 6 hours (P1), and SCI induction with termination at 12 hours (P2). S100β expression was analyzed using one-way ANOVA and post-hoc Tukey HSD tests.

Results: Significant differences in S100β expression were found among the groups (p = 0.006). Post-hoc analysis showed a significant increase in S100β expression in the P1 group compared to the control group (p = 0.005). No significant differences were observed between the control and P2 groups (p = 0.480) or between P1 and P2 (p = 0.077).

Conclusion: S100β expression increases significantly 6 hours after SCI but decreases by 12 hours, approaching control levels. This suggests that astrocyte activity and cell destruction are most prominent early after injury, with recovery occurring over time.

S100β Protein

Spinal Cord Injury (SCI)

Astrocytes

Histopathological Expression

Rattus norvegicus

Spinal Cord Injury (SCI) refers to any direct or indirect injury that disrupts sensory, motor, and autonomic functions.¹ According to the World Health Organization (WHO), the global incidence of SCI ranges from 40 to 80 cases per million people annually. SCI is more prevalent in men than women, with a ratio of 2:1, and is most commonly seen in adolescents and young adults (20–29 years old). In Indonesia, data on the incidence of SCI is still being researched.² A study at Fatmawati Central General Hospital in Jakarta recorded 104 cases of SCI, with 37 cases being traumatic and 67 cases non-traumatic. The most common causes of traumatic SCI were motor vehicle accidents and falls from heights, while non-traumatic SCI was mainly due to infections and neoplasms.³

S100β protein is a low molecular weight (9–13 kDa) Ca2+-binding protein primarily found in glial astrocytes of the central nervous system.⁴ Astrocytes secrete s S100β and plays a role in calcium homeostasis, acting as both a neuroprotective and neurotrophic agent in the central nervous system. As a neuroprotective agent, S100β can inhibit cell death and mitochondrial failure in glucose-deprived conditions by increasing Ca2 + concentrations. As a neurotrophic agent, S100β stimulates neurite growth and astrocyte proliferation, thereby enhancing neuronal function. S100β is specifically found in the cytoplasm and nucleus, with less than 1% of this protein being secreted.⁵

Serum and cerebrospinal fluid levels of S100β have been reported to increase significantly in cases of brain and spinal cord injuries.^4,6,7 A meta-analysis of six experimental studies on animal models found that S100β levels have significant diagnostic value for SCI. This marker increases within 6 hours post-injury and remains elevated up to 24 hours after trauma. Beyond 24 hours, S100β levels decrease to those seen in the absence of spinal cord injury.⁸ There has not been any previous studies that report of S100β histopathological expression on SCI.

Due to its significant rise in conditions of brain and spinal cord injuries within 6 hours post-trauma, S100β can be used as a marker for acute spinal cord injury. The pathophysiology of SCI begins immediately after trauma and can progress to chronic injury over time. Additionally, SCI often accompanies traumatic injuries, such as those from traffic accidents and falls. Patients can experience permanent neurological deficits depending on the location and extent of the injury. This also affects the patient's survival rate, making it crucial to identify and manage SCI within the first few hours post-trauma.⁹

Proper and immediate management of SCI is essential, as errors and delays can increase the risk of secondary injuries, thereby raising morbidity and mortality rates and worsening patient prognosis. Determinants of SCI prognosis include emergency management before reaching a healthcare facility, the transportation system to the healthcare facility, and the quality of care provided at the healthcare facility.¹¹

2.1 Study Population

This study is an experimental research with true experimental design conducted in vivo using a post-test-only randomized control group design. There are three groups in this study: one control group without treatment (K), one treatment group induced with moderate Spinal Cord Injury using a 35 g clip load and terminated after 6 hours (P1), and another treatment group induced with moderate Spinal Cord Injury using a 35 g clip load and terminated after 12 hours (P2).

The inclusion criteria for this study are: 1) Sprague-Dawley rats; 2) weighing 250–300 grams; 3) 3 months old; 4) male. Experimental animals showing signs of illness will be excluded from this study.

2.1 Data Collection

The sample size calculation was performed using Federer's formula. There are three treatment groups in this study: the control group without treatment (K), the treatment group induced with moderate SCI and terminated after 6 hours (P1), and the treatment group induced with moderate Spinal Cord Injury and terminated after 12 hours (P2). The total number of subjects required for this study is 30.

2.2 Data Analysis

The One-Way ANOVA or Kruskal-Wallis test was selected due to its numerical data and involves more than two independent groups. Data analysis was conducted using Statistical Product and Service Solutions (SPSS) version 23.00 for Windows.

The highest average expression of S100β tissue was in group P1 (treatment with vascular clip with a clamping force of 35 g for 1 minute then terminated after 6 hours), which was 42.8445; while the lowest average expression of S100β tissue was in group K (without treatment), which was 19.7528. (Figs. 1 and 2).

The highest average expression of S100β tissue was observed in group P1 (treated with a vascular clip with a clamping force of 35 g for 1 minute, then terminated after 6 hours), with a mean value of 42.8445. In contrast, the lowest average expression of S100β tissue was found in group K (untreated), with a mean value of 19.7528 (Figs. 1 and 2). According to Table 1, the p-values for each group were greater than 0.05, indicating that the level of significance for each group exceeded the predetermined threshold. Thus, the requirements for conducting a One-Way ANOVA were met. The One-Way ANOVA test yielded a p-value of 0.006 (p < 0.05). Based on these results, it can be concluded that there is a significant difference in S100β tissue expression among the groups of rats.

Table 1

S100β Tissue Expression Normality Test Results
Group	N	P
K	10	0,687
P1	10	0,073
P2	10	0,770

Subsequently, a Tukey HSD test was performed to determine the significance of the differences in S100β tissue levels in spinal cord tissue among the groups. The Tukey HSD test results between groups K and P1 showed a p-value of 0.005 (p < 0.05), indicating a significant difference in S100β expression scores in spinal cord tissue between K and P1. Thus, it can be concluded that spinal cord compression causes a significant increase in S100β tissue expression on spinal cord tissue 6 hours post-compression. The Tukey HSD test results between groups K and P2 showed a p-value of 0.480 (p > 0.05), indicating no significant difference in S100β tissue expression scores on spinal cord tissue between K and P2. Thus, it can be concluded that spinal cord compression does not cause a significant increase in S100β tissue expression on spinal cord tissue 12 hours post-compression. The Tukey HSD test results between groups P1 and P2 showed a p-value of 0.077 (p > 0.05), indicating no significant difference in S100β expression scores in spinal cord tissue between P1 and P2, despite a decrease in S100β expression from 42.8445 in group P1 to 27.5959 in group P2. As can be seen in Table 2, these statistical findings are summarized, further supporting the interpretation of the results.

Table 2

Tukey HSD Test Results of S100β Tissue Expression in Rat Spinal Cord Tissue
Group	Compared to Group	Differences	Sig.
K	P1	-23,09165	0,005
P1	P2	15,24860	0,005
P2	K	7,84305	0,480

The results of this study indicate that spinal cord compression can cause a significant increase in S100β tissue expression on spinal cord tissue 6 hours post-compression. These findings are consistent with several previous studies. A research by Marquardt et al.¹² demonstrated an increase in serum S100β tissue levels 6 hours post-injury in 30 rats subjected to spinal cord injury via contusion trauma, compared to control rats that only underwent laminectomy.¹²

A meta-analysis involving 7 research articles reported an increase in serum S100β tissue levels within the first 6 hours post-trauma (SMD = 3.8; 95% CI 2.6–5.1; p < 0.0001), as well as cerebrospinal fluid (CSF) S100β levels within the first 6 hours post-trauma (SMD = 5.8; 95% CI 3.6-8.0; p < 0.0001). This meta-analysis also analyzed serum S100β levels in severe spinal cord injury (SMD = 3.4; 95% CI 1.6–5.4; p < 0.0001) and moderate spinal cord injury (SMD = 1.6; 95% CI 0.8–2.4; p < 0.0001), finding that S100β levels were higher in severe cases compared to moderate cases.⁸

This study reports S100β expression tissue in moderate spinal cord injury, represented by a vascular clip with a clamping force of 35 g.¹³ However, S100β levels from CSF in moderate (SMD = 4.1; 95% CI 2.4–5.8; p < 0.0001) and severe (SMD = 4.1; 95% CI 2.1–6.2; p < 0.0001) spinal cord injury do not differ significantly. While both studies used animal models, this study assessed S100β through histopathological analysis, unlike the aforementioned studies that evaluated serum S100β levels.

Another study on humans investigated the prognosis of S100β tissue in spinal cord injury at 24 and 48 hours post-injury in serum and CSF. A significant positive correlation was found between CSF S100β levels and complete spinal cord injury (p = 0.002) within the first 24 hours post-trauma. The cutoff value for CSF S100β was 342.18 ng/dL, with a sensitivity of 100% and specificity of 64%.¹⁴ However, Haddadi et al.¹⁴ did not assess S100β levels at 6 and 12 hours and only found significant correlations in CSF samples. Nonetheless, Haddadi et al.'s study was conducted on human subjects.

An increase in S100β occurs when trauma disrupts the blood-brain barrier (BBB), typically due to central nervous system trauma, leading to elevated S100β levels.¹⁵ Most S100β protein, a calcium-binding protein, is found in the cytoplasm of glial cells. Since the BBB is impermeable to this protein, it is not found in serum and CSF unless there is central nervous system injury causing BBB disruption, which results in increased serum S100β levels.¹⁶ Various experimental studies have shown that serum and CSF S100β levels rise rapidly shortly after injury and then gradually decrease to normal levels within a few days. Additionally, S100β has been investigated as a predictor of early spinal cord damage, directly reflecting the severity of spinal cord injury. Significant changes in serum S100β levels are also observed in patients with spinal fractures and spinal cord injuries. S100β protein could be a useful tool for early detection of spinal cord involvement and neurological outcomes within 6 months post-spinal cord injury.¹⁷

There was no significant difference in S100β expression scores on spinal cord tissue between the control group (K) and the group treated with spinal cord compression terminated at 12 hours (P2). Spinal cord compression did not cause a significant increase in S100β expression scores on spinal cord tissue 12 hours post-compression. These results differ from previous studies. A meta-analysis involving 7 research articles reported an increase in serum S100β levels within the first 12 hours post-trauma (SMD = 2.7; 95% CI 0.5–4.9; p = 0.018), as well as an increase in CSF S100β levels within the first 12 hours post-trauma (SMD = 6.5; 95% CI 3.7–9.3; p < 0.0001).⁸ Although this meta-analysis observed a decline in serum and CSF S100β levels 12 hours post-spinal cord injury, the decrease was not statistically significant. In contrast, this study reported that S100β expression in spinal cord tissue 12 hours post-injury was relatively similar to pre-injury levels. The discrepancy in findings may be due to the different media used for assessing S100β levels. The meta-analysis measured S100β levels from serum and CSF, while this study assessed S100β expression from spinal cord histopathological samples. The elevated serum and CSF S100β levels might originate from other tissues besides the spinal cord, as elevated S100β levels have also been found shortly after running¹⁸, in bone fractures, thoracic contusions without fractures, burns¹⁹, parkinson²⁰, and ischemic stroke.²¹

There was no significant difference in S100β expression scores on spinal cord tissue between groups P1 and P2, despite a decrease in S100β expression from 42.8445 in group P1 to 27.5959 in group P2. This decline in S100β expression post-spinal cord injury is consistent with previous studies. A meta-analysis by Faridaalee and Keyghobadi Khajeh⁸ found that S100β levels were very high during the first 6 hours after injury (SMD = 3.8; 95% CI: 2.6–5.1; p < 0.0001) and within the first 12 hours after injury (SMD = 2.7; 95% CI 0.5–4.9; p = 0.018). However, over time, serum protein levels decreased, and after more than 24 hours, the values were almost identical to the control group without spinal cord injury (SMD = 0.4; 95% CI: 1.2-2.0; p = 0.65). The decline in S100β indicates a reduction in astrocyte glial cell destruction over time until the rate of destruction ceases.²²

This study only investigated S100β levels in the control group and in groups with spinal cord injury at 6 and 12 hours post-trauma, not extending to 24 or 48 hours post-trauma. Moreover, the study was limited to animal models. Clinical trials in humans at 6 and 12 hours post-trauma are necessary.

There is a difference in S100β expression on the spinal cord tissue of the Sprague-Dawley rat (Rattus norvegicus) model with spinal cord injury compared to those without injury.

Consent TO PUBLICATIONS

Informed consent was obtained from all individual participants included in the study. For participants under the age of 18 or those unable to provide consent, consent was obtained from their legal guardians.

Consent TO PARTICIPATE

Informed consent was obtained from all individual participants included in the study. Prior to participation, each participant was provided with a detailed explanation of the study's purpose, procedures, potential risks, and benefits. Participants were informed of their right to withdraw from the study at any time without any consequences. Written consent was obtained from each participant, ensuring their voluntary involvement and confirming their understanding of the research process.

DATA AVAILABILITY STATEMENT

The data generated from the study titled *"Comparative Analysis of S100β Expression on Spinal Cord Tissue of Injured vs. Uninjured Rat Models (Rattus Norvegicus Sprague-Dawley)"* will be made available upon reasonable request. All relevant datasets, including raw and processed data related to S100β expression analysis, will be stored in a secure institutional repository. Access to the data will be granted to qualified researchers for the purpose of verification or further study, following institutional guidelines and upon approval from the corresponding author.

FUNDING

This research, titled *"Comparative Analysis of S100β Expression on Spinal Cord Tissue of Injured vs. Uninjured Rat Models (Rattus Norvegicus Sprague-Dawley),"* did not receive any specific funding from public, commercial, or not-for-profit agencies. All research expenses were covered solely by the authors.

Authors’ Contributions

The authors declare that they have no competing interests related to this study. All authors have read and approved the final manuscript.

Ethical approval

This study, titled *"Comparative Analysis of S100β Expression on Spinal Cord Tissue of Injured vs. Uninjured Rat Models (Rattus Norvegicus Sprague-Dawley),"* will be conducted in accordance with ethical guidelines for animal research, ensuring humane treatment of the animals, minimizing suffering through the use of anesthesia and analgesia, and adhering to the principles of Replacement, Reduction, and Refinement (3Rs). Approval from the Institutional Animal Care and Use Committee (IACUC) will be obtained prior to the study's commencement.

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No competing interests reported.

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Comparative Analysis of S100β Expression on Spinal Cord Tissue of Injured Vs. Uninjured Rat Models (Rattus Norvegicus Sprague-Dawley)

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Abstract

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1. Introduction

2. Material and Methods

2.1 Study Population

2.1 Data Collection

2.2 Data Analysis

3. Results and Discussion

4. Conclusion

Declarations

References

Additional Declarations

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