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. 2026 Sep 19;21:874–883. doi: 10.1016/j.ibneur.2026.09.014

Penetrating spinal cord injury transiently inhibits responsiveness to touch in mice

Taketo Otani a, Shunsuke Nakamura b, Tokue Mieda c,⁎, Kazuhiro Nakamura a,⁎
PMCID: PMC13634452  PMID: 42831051

Abstract

Spinal cord injury (SCI) is a devastating mechanical trauma of the spinal cord. We recently showed that penetration of fine needles from dorsal to ventral regions in the spinal cord disturbed spontaneous locomotion in mice. However, it has not been verified if penetrating SCI at the dorsal side of the spinal cord, responsible regions for sensory functions, changes movements after sensory stimulation. In this study, penetration into the dorsal midline or dorsolateral regions was separately applied to mice. Using these mice, we evaluated withdrawal movements after tactile and thermal stimulations as well as locomotion. Penetrating SCI at midline and dorsolateral regions exhibited normal Basso Mouse Scale for Locomotion (BMS) and beam walking test scores. In addition, locomotion in open field was not affected in these mice. Also, we showed that responses after thermal stimulation on hot plate was not changed in mice with midline and dorsolateral penetrations. However, paw withdrawal after both static and dynamic touches were transiently inhibited in mice after both midline and dorsolateral penetrations. This model is a valuable tool for examining the functional and pathological consequences of damage to specific, discrete regions or tracts within the spinal cord.

Keywords: mouse, penetration, spinal cord injury

1. Introduction

Spinal cord injury (SCI) is a devastating mechanical trauma of the spinal cord. The main cause of SCI is traffic accident (Alizadeh et al., 2019). Since the accident applies momentary pressure broadly to the entire spinal cord, the SCI patients after traffic accident likely suffer from motor, sensory and bladder dysfunctions (Fiore et al., 2004). Rat and mouse models for contusion SCI is well established (Akhtar et al., 2008, Cheriyan et al., 2014, Maiman et al., 1983) and have been used to evaluate therapeutic effects of agents.

On the other hand, penetration of fine-tipped objects into the spinal cord (penetrating SCI) results in injuries in local areas in the spinal cord. Penetrating SCI is mainly divided into missile and non-missile penetration. Gunshots can cause missile penetrating SCI in humans (Bin-Alamer et al., 2022). Regarding rare non-missile penetrating SCI (Abdulqader et al., 2022), knife stabs are the most common cause (Bin-Alamer et al., 2022). Since length of stay of penetrating SCI is long (Avila et al., 2021), knowledge on penetrating SCI should be accumulated using animal models.

Recently, mouse models of penetrating SCI were generated (Shirota et al., 2024). In the mice, fine needles were bilaterally inserted from the dorsal side to ventral side. The insertion was applied to medial (0.1 mm from the midline) and lateral (0.4 mm from the midline) regions of the spinal cord. Using the mice, spontaneous locomotion was investigated because the penetrated ventral regions controls movement (Shirota et al., 2024). Both mice with medial and lateral penetrations showed essentially no alterations in beam walking (Shirota et al., 2024). In contrast, overground walking was impaired in the both mice (Shirota et al., 2024), as evidenced by Basso Mouse Scale for Locomotion (BMS)(Basso et al., 2006). Similarly, open-field test clarified that movement duration, moving speed and/or number of movement in the open field were changed in the two mice (Shirota et al., 2024). These results indicate that penetration into the ventral regions in the spinal cord disturbed spontaneous locomotion in mice. However, it has not been verified if penetrating SCI specifically at the ventral side of the spinal cord, responsible regions for sensory functions, changes movements after sensory stimulation.

There are not so many reports regarding penetrating SCI in humans (Beer-Furlan et al., 2014, Deguchi et al., 2022, Dogan et al., 2008, Meena et al., 2016, Rabiu et al., 2011, Ye et al., 2010). Symptoms of the patients are motor and/or sensory disturbance and some cases presented Brown-Sequard syndrome characterized by ipsilateral motor impairment and loss of proprioception and contralateral loss of pain and temperature sensation. In some cases, no improvement in motor and sensory disturbance was seen after emergency surgery. In other cases, motor deficits were recovered without improvement of sensory deficit after surgery. These observations indicate that sensory deficits after penetrating SCI might be refractory to surgery. From the context, it is particularly important to determine the functional and pathological consequences of damage to specific, discrete regions or tracts within the spinal cord.

Among modality of sense, we focused on responsiveness to touch as the initial model case because neural circuit underlying the responsiveness is well studied. In the current study, we specifically applied penetration to the dorsal side of the spinal cord. Cutaneous sensory information is conveyed via multiple regions such as the dorsal horn, dorsal columns and spinothalamic tract. Therefore, penetration into the midline and dorsolateral regions of the spinal cord was separately applied to mice. These penetrations likely affect these regions. Using these mice, we evaluated withdrawal movements after tactile and thermal stimulations. We also investigated locomotion using BMS, beam walking test scoring and open field test.

2. Materials and methods

2.1. Mice

ICR mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and kept in specific pathogen-free room where the temperature was kept at 23°C and lighting was 12 h every day. We followed NIH guidelines of mouse experiments and made our best efforts to reduce suffering of mice. The number of mice used for the experiments was minimum to obtain reliable data. Mouse experiments were approved by the Animal Resource Committees of Gunma University (Approval number, 25–009).

2.2. Spinal cord penetrating injury

Spinal cord inside the T8 or T9 vertebral spine was exposed. Then, the mouse was fixed on stereotaxic instrument. Thirty three-gauge needle (Hamilton company, Reno, NV) was vertically inserted into the spinal cord at midline (0.6 mm deep) or at dorsolateral sites (0.6 mm from the midline, 0.6 mm deep bilaterally)(Fig. 1). When penetrating injury was given at both dorsolateral sites, the left side was first injured and then needle was inserted in the right side where the needle was inserted for 120 s. Base of the 33-gage needles was approximately 0.21 mm in diameter. No infection and bladder and rectal disorders were found during observation period.

Fig. 1.

Fig. 1

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Insertion sites of needles for penetrating spinal cord injuries. Schematic illustration reveals size and insertion sites of needle. Operation was conducted without inserting needles in sham mice as a control (A). Mice with two different types of spinal penetrating injury were subjected to behavioral tests. In the first group, a needle (0.21 mm in diameter) was inserted at the midline of the spinal cord (B). In the second group, the needle was bilaterally inserted in the dorsolateral regions of the spinal cord (0.6 mm from the midline)(C). In the two groups, depth of the needle was identical (0.6 mm from the surface of meninges).

2.3. Behavioral tests

Mice at 2–3 months old were randomly divided into groups for behavioral tests. The tests were initially performed before SCI. Then, the tests were also done 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks after SCI. We prepared 2 sets of mice. Using the first set of mice, multiple behavioral tests were done in the following order; open field test, beam-walking test scoring, BMS and hot plate test.

For BMS (Basso et al., 2006), mouse was allowed to walk in an open field (60 cm×120 cm) for 2 min. The scoring was done according to visual inspection regarding parameters such as paw and tail positions, ankle movement, and trunk instability and coordination. When the score was different between the right and left hindlimbs, an averaged value was applied.

Detailed protocol of beam-walking test scoring was previously described (Ito et al., 2022). Briefly, walking appearance of the mouse on an elevated round horizontal beam 100 cm long and 11 mm in diameter was scored. Mouse was initially trained to traverse the narrow beam. Then, the score was determined whether the mouse retains for 2 min, moves forward, reaches the goal and slips on the beam. Position of hindlimbs on the beam was also scored. We tested 3 times and the highest point was used for each parameter to calculate the total score.

Open field test was done for 10 min using an apparatus with square field (50 × 50 cm) surrounded by walls (50 cm high) with an open top (O’HARA & Co., Ltd., Tokyo, Japan). Total walking distance, duration of movement during test session, distance per movement, duration per movement and number of episode of movement were measured.

Hot plate test was done using an apparatus (O’HARA & Co., Ltd., Tokyo, Japan) having heated surface (52.5 °C). Lifting or licking of hindpaw and jumping were used as the time elapsed to paw withdrawal (Meymandi and Keyhanfar, 2012, Xiong et al., 2021).

Paw withdrawal after touch was measured using the second set of mice because stimulation of a type of sensory modality (pain) might interfere with sensitivity of different kind of sensory modality (touch). Mice were allowed to stand on a wire mesh (6.5 ×6.5 mm). Then, a soft paintbrush (16 ×6 mm) was used to stimulate the hindpaw from behind of the mesh. The paintbrush was made of nylon and aluminum (Daiso Industries Co., Ltd., Hiroshima, Japan). First, gentle stroking of hindpaw was applied from heel to toe as the dynamic touch. Then, the paintbrush was perpendicularly applied to the plantar surface of the hindpaw as the static touch (Paixao et al., 2019). Touches were applied alternately left and right 10 times with an interval of 30 s and frequency of the paw withdrawal after touches was counted as described (Paixao et al., 2019). After the measurement at 4 weeks, open field test was conducted once to evaluate correlation between number of withdrawal after dynamic and static touches and total walking distance in open field.

2.4. Histology

After transcardiac perfusion with 4% paraformaldehyde in PBS, dissected spinal cord covered with spine was postfixed with the same fixative solution overnight at 4°C. Then, the block was subjected to decalcification with G-Chelate Quick (GenoStaff, Tokyo, Japan) for 2 days. After neutralization for a day, the block was embedded in paraffin. Transverse spinal sections 3 µm in thickness were prepared every 300 μm. The sections of rostral and caudal ends were approximately 1000 μm from the injury site. Before staining, sections were deparaffinized with xylene followed by rehydration with graded alcohols. For HE staining, the sections were stained with hematoxylin and eosin. For Luxol fast blue staining, the sections were stained with 0.1% Luxol fast blue solution (MUTO PURE CHEMICALS CO., LTD, Tokyo, Japan) at 65 °C overnight. Then, the sections were differentiated in 0.05% Lithium carbonate and 70% ethanol. The stained sections were treated with graded alcohol and xylene. Finally, the sections were mounted. Images were taken using a Nikon ECLIPSE 80i microscope (NIKON CORPORATION, Tokyo, Japan).

2.5. Statistical analysis

The values were expressed as the mean ± SE. Results of behavioral tests were statistically analyzed using Friedman test followed by Scheffe test because normality was not met. Correlation between number of withdrawal after dynamic and static touches and total walking distance in open field test was estimated by Spearman’s rank correlation coefficient. p values less than 0.05 were considered as statistically significant.

3. Results

3.1. Establishment of mouse models for penetrating SCI at dorsal side with fine needles

To determine whether penetrating injuries in the dorsal regions affect movements after sensory stimulations, the needle should be inserted to penetrate the regions responsible for integration between sensory and motor functions in mouse. Location of the descending tracts in the spinal cord is different between human and rodents (Kjell and Olson, 2016, Watson and Harrison, 2012). Apparent difference between human and rodents is seen in the location of the corticospinal tract (Kjell and Olson, 2016). In humans, the tract occupies large areas in the lateral white matter. In contrast, majority of the tract is located in the dorsal midline regions in rat and mouse spinal cord (Kjell and Olson, 2016, Watson and Harrison, 2012). Although the corticospinal tract mainly controls voluntary movement, responses after light touch is also modulated by activity of the corticospinal tract (Liu et al., 2018). Likewise, the dorsal column located in the dorsal midline region of the spinal cord is a relay point of transmission route of sensory information from the periphery to the brain (Turecek et al., 2022). Thus, we inserted needles into the midline region of dorsal spinal cord inside the T8 or T9 vertebral spine (Fig. 1B). The depth of needles was 0.6 mm from the surface of meninges to apply pressure to the dorsal column and the corticospinal tract.

The dorsal horn and spinothalamic tracts are also the relay points of sensory information. The dorsal horn occupies broad area in the dorsal regions and the spinothalamic tracts are located in the lateral and ventral regions in mouse (Watson and Harrison, 2012). Therefore, the needle was also bilaterally inserted in the dorsolateral regions of the spinal cord (0.6 mm from the midline). Depth of the needle was 0.6 mm (Fig. 1C). The insertion is expected to apply pressure to broad area of the lateral end of the dorsal horn and also part of the spinothalamic tracts. We also prepared sham mice to which operation was applied without needle insertion (Fig. 1A).

3.2. No change in motor function after penetrating SCI at dorsal side

We first assessed motor functions. BMS score was maximum (score of 9) at almost all time points in all mice (Fig. 2A). This result suggests that overground walking was not essentially affected by the penetrating injuries. Similarly, the beam walking test scores (Ito et al., 2022) after penetrating SCIs were also maximum score (score of 7) at almost all time points in all mice (Fig. 2B). Thus, beam walking was not affected by the insertion as well.

Fig. 2.

Fig. 2

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BMS and beam walking test scores in mice after penetrating spinal cord injuries. BMS (A) and beam walking test (B) scores were shown in sham (n = 8, male 4, female 4), midline penetrating SCI (n = 9, male 3, female 6) and dorsolateral penetrating SCI (n = 11, male 5, female 6) mice 1 day (d), and 1, 2, 3 and 4 weeks (w) after injuries. The scores before operation (pre) were also shown. Each error bar represents SE.

We then performed open field test to evaluate overground walking. In the test, the mouse was allowed to freely walk in open field. The values after SCI was compared to those before SCI. Total walking distance (Fig. 3A), total movement duration (Fig. 3B), distance per movement (Fig. 3C), duration per movement (Fig. 3D) and number of movement episode (Fig. 3E) were not changed after penetrating injuries in all groups. Collectively, no alterations were found in overground walking.

Fig. 3.

Fig. 3

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Open field test in mice after penetrating spinal cord injuries. Total walking distance (A), total movement duration (B), distance per movement (C), duration per movement (D) and number of movement (E) in open field were shown in sham (n = 8, male 4, female 4), midline penetrating SCI (n = 9, male 3, female 6) and dorsolateral penetrating SCI (n = 11, male 5, female 6) mice 1 day (d), and 1, 2, 3 and 4 weeks (w) after injuries. The values before operation (pre) were also shown. Each error bar represents SE.

3.3. Altered responsiveness to light touch after penetrating SCI at dorsal side

We next performed behavioral tests to assess hindlimb withdrawal after thermal or tactile stimulation. Thermal stimulation was applied using hot plate test. The mouse was put on heated plate and measured the time when the mouse withdraws their foot. In all groups, no differences were obtained (Fig. 4). Finally, we assessed hindlimb withdrawal after light touch. The mouse underwent gentle stroking of hindpaw (dynamic touch) with paintbrush 10 times and frequency of the paw withdrawal was counted. In sham group, the frequency was not significantly altered after operation compared to pre (Fig. 5A). However, the significant differences were found in other groups. In mice given penetrating SCI at the midline, the number of withdrawal decreased 1 and 2 weeks after SCI compared to that before SCI (Friedman test followed by Scheffe test, p < 0.001 and p = 0.02 at 1 and 2 weeks, respectively). Then, the difference disappeared at 3 and 4 weeks compared to that before SCI (p = 0.36 and 0.25 at 3 and 4 weeks, respectively)(Fig. 5A). In mice with dorsolateral penetration, the numbers decreased from 1 to 3 weeks after SCI (Friedman test followed by Scheffe test, p = 0.02, 0.04 and 0.04 at 1, 2 and 3 weeks, respectively). At 4 weeks, the difference was no longer seen (p = 0.36)(Fig. 5A).

Fig. 4.

Fig. 4

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Performances of hot plate test in mice after penetrating spinal cord injuries. Latency of reaction on hot plate in sham (n = 8, male 4, female 4), midline penetrating SCI (n = 9, male 3, female 6) and dorsolateral penetrating SCI (n = 11, male 5, female 6) mice 1 day (d), and 1, 2, 3 and 4 weeks (w) after injuries. The test was also done before operation (pre). Each error bar represents SE.

Fig. 5.

Fig. 5

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Responsiveness to light touch after penetrating spinal cord injuries. (A, B) Number of reaction after dynamic (A) and static (B) touches in sham (n = 5. male 1, female 4), midline penetrating SCI (n = 7, male 4, female 3) and dorsolateral penetrating SCI (n = 6, male 4, female 2) mice. The tests were done before operation (pre) and 1 day (d), and 1, 2, 3 and 4 weeks (w) after operation. Each error bar represents SE. The value at each time point after operation was compared with that of pre using Friedman test followed by Scheffe test. *p < 0.05, **p < 0.01. (C, D) Comparison of dynamic (C) and static (D) touch test performances among the 3 groups. Values before SCI in all groups were expressed as 100% and the values relative to that before SCI were plotted. (E, F) Correlation between responsiveness to light touch and total walking distance in open field at 4 weeks after operation. Total walking distance in open field (x-axis) and number of reaction to dynamic (E) or static (F) touch (y-axis) were plotted in the graph.

We next examined responses after static touch, in which paintbrush was perpendicularly applied to the plantar surface without moving. The touch was applied 10 times and the frequency of the paw withdrawal was counted. Number of response of sham mice after static touch was significantly decreased 1 week after operation compared to pre (Friedman test followed by Scheffe test, p = 0.03). However, the difference disappeared at 2 weeks (p = 0.22). Then, there were no differences at 3 and 4 weeks (p = 0.75 and 0.87, respectively)(Fig. 5B). Therefore, we focused on the response after static touch 2–4 weeks after SCI.

In mice given penetrating SCI at the midline, the number of withdrawal was smaller at 2 weeks after SCI compared to that before SCI (Friedman test followed by Scheffe test, p = 0.02)(Fig. 5B). The difference was not seen at 3 and 4 weeks (p = 0.13 and 0.43, respectively). In mice given dorsolateral penetration, no difference was observed at 2 weeks (p = 0.17). However, significant difference was seen at 3 weeks (p = 0.03). Then, the difference disappeared at 4 weeks (p = 0.55)(Fig. 5B). To facilitate comparison between groups, values before SCI in all groups were expressed as 100% and the values relative to that before SCI were plotted in Fig. 5C, D. Numbers of response of the two SCI mice after dynamic (Fig. 5C) and static (Fig. 5D) touches were smaller than those of sham mice as a whole. Taken together, penetrating injuries at the midline and dorsolateral regions transiently led to decreased number of response after light touch. However, the reduction was spontaneously recovered.

Since general hindlimb motor function potentially affects responsiveness to light touch, we then checked correlation between total walking distance in open field test and number of reaction after dynamic and static touches in SCI mice. Open field test was done after the final light touch at 4 weeks. Spearman’s rank correlation coefficient was 0.26 (p = 0.29) between number of response to dynamic touch and total walking distance (Fig. 5E) and 0.14 (p = 0.58) between that of static touch and total walking distance (Fig. 5F). These results do not indicate that hindlimb motor function dominantly affected responsiveness to light touch. Thus, it is unlikely that changes in motor function itself affected the results of withdrawal after touches.

3.4. Cavity formation in part of penetrating SCI mice

Finally, we examined histological changes in mice after penetrating SCI using mice 1 week after penetrating SCI because 1 week is the time when dynamic touch test performance is affected in both midline and dorsolateral penetrating SCI mice. Many cavities were found in the dorsal column after midline penetration in two out of four mice but not in sham mice (Fig. 6A, B, F). The cavities were seen over a distance of at least 900 µm along the rostral-caudal axis of the spinal cord. The cavities were also found in the dorsolateral white matter of midline penetrating SCI mice (Fig. 6B, G). There were no cavities in the remaining two mice after midline penetrating SCI (Fig. 6C). Likewise, cavities were detected in the dorsal column (Fig. 6D, H) and the dorsolateral white matter (Fig. 6D, I) after dorsolateral penetration in two out of three mice. The cavities were not seen in the remaining mouse after dorsolateral penetrating SCI (Fig. 6E). Infiltration of inflammatory cells was detected around the cavities (Fig. 6F-I). Cluster of inflammatory cells was more apparent in the dorsal column than the dorsolateral white matter. Majority of the cells were lymphocytes.

Fig. 6.

Fig. 6

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Histology of the spinal cord after penetrating spinal cord injuries. Sham (A, J, M) and midline (Mid)(B, C, F, G, K, N) and dorsolateral (DL)(D, E, H, I, L, O) penetrating SCI mice were processed for HE (A-I) and Luxol fast blue (J-O) staining. Small (A-E) and large (F-O) magnification images are shown. Arrows and arrowheads indicate cavities and cluster of inflammatory cells, respectively. Lower myelin density areas are marked with asterisks. DC, dorsal column; DLWM, dorsolateral white matter; DR, dorsal root. Scale bars, 100 µm.

We then checked density of myelin in mice with cavities and inflammatory cells by Luxol fast blue staining. Penetration into the dorsal spinal cord at midline led to lower density of myelin in the dorsal column (Fig. 6K) compared with sham mice (Fig. 6J). The lower density was not so apparent in the dorsolateral white matter of midline penetrating SCI mice (Fig. 6N) compared with sham mice (Fig. 6M). Similarly, penetration into the dorsolateral region induced lower density of myelin in the dorsal column (Fig. 6L) but not in the dorsolateral white matter (Fig. 6O) compared with sham mice (Fig. 6J, M). However, density of myelin in the dorsal root was not altered in midline and dorsolateral penetrating SCI mice (Fig. 6M-O).

4. Discussion

In the present investigation, we gave penetrating SCI to regions including the midline corticospinal tract in mice and found no alterations in BMS, beam walking test score and all indices of open field test. The corticospinal tract, an axon bundles projecting from the motor cortex in the brain, transmits directive from the brain to muscles. The tract passes through the dorsal midline regions in rat and mouse spinal cord (Kjell and Olson, 2016, Watson and Harrison, 2012). Degree of contribution to voluntary movement of the corticospinal tract is different between human and rodents. Bilateral lesions of the corticospinal tract largely affected walking in humans (Nathan, 1994), whereas, lesions of the corticospinal tract had a less severe effect on gait in rats. Instead, other descending pathways that are responsible for movements are crucial for gait (Filli et al., 2014, Kjell and Olson, 2016, Schucht et al., 2002). The current results indicate that other descending pathways likely compensated for the functions of partially injured corticospinal tract in mice.

In our previous paper (Shirota et al., 2024), bilateral penetration was applied to sites 0.1 mm lateral from the midline and these mice exhibited altered BMS score and values in multiple indices of open field test. In contrast, no changes were observed in the test in mice given penetration into the midline precisely in the present study. The needle deeply penetrated both the ventral region and the dorsal region in the previous study (Shirota et al., 2024). By contrast, the penetration reached the corticospinal tract in the dorsal regions but not the ventral regions in this study. Moreover, needle insertion at 0.1 mm lateral from the midline in the previous study penetrates inside the corticospinal tract. In the case of the midline insertion in this study, the needle attaches medial ends of the corticospinal tract and likely gives pressures to neurons in the corticospinal tract. Intact corticospinal tract neurons located distantly from the needle likely compensated for the injured neurons and eventually led to normal hindlimb appearance during overground walking, as assessed by BMS and open field tests. Collectively, subtle difference in sites of penetration near the midline of spinal cord results in distinct locomotion pattern in mice.

We found cavity formation in both dorsal columns and dorsolateral white matter of midline penetrating SCI mice. As a similar histological change after SCI, vacuolar formation after SCI was previously reported (Sakti et al., 2024). In detail, histological examination of the dogs given balloon compression-induced SCI revealed vacuolar formation at intra-, supra- and/or infra-lesion sites. This observation is in line with our findings that the cavity formation was also recognized in regions distant from the needle insertion sites.

As the cause of cavity formation in this study, the midline penetration might injure the central canal of spinal cord and induce leakage of cerebrospinal fluid (CSF), which likely results in fluid accumulation inside the spinal cord as seen in CSF-filled cyst formation in patients with syringomyelia (Heiss et al., 2019). In this context, the dorsolateral penetration might broadly injure the arachnoid, leading to leakage of CSF from the subarachnoid space and cavity formation in the spinal cord. We found lower density of myelin in regions close to the cavities in the dorsal columns but not so apparent in the dorsolateral white matter in SCI mice. The difference is likely due to degree of inflammatory cell infiltration because that was higher in the dorsal columns than in the dorsolateral white matter.

Despite cavity formation and lower density of myelin in the dorsal columns having the corticospinal tract in the penetrating SCI mice, motor dysfunction was not recognized. Likewise, results of hot plate test were not altered although cavity formation was also seen in the dorsolateral white matter of SCI mice. Thus, these morphological changes themselves might not be the major cause of dysfunction in touch tests. In line with this hypothesis, dynamic touch test performance became normal 4 weeks after contusion SCI in spite of massive demyelination (Kakuta et al., 2019). Collectively, functional changes such as neural transmission after dorsal penetration might additively contribute to the dysfunctions because cavity formation was not seen in almost half of penetrating SCI mice in this study. The cavity formation might depend on extent of invasion of the central canal wall by needle penetration. Consistently, inter-individual difference in vacuolar formation was also found in SCI dog (Sakti et al., 2024).

In the present investigation, we showed that withdrawal after thermal stimulation using hot plate was not changed in mice with midline and dorsolateral penetrations. However, withdrawal after light touch was inhibited in mice after midline and dorsolateral penetrations. The difference between the results of two tests might be due to difference in the transmission pathways among modality of sensory information. Innocuous touch signals and nociceptive and thermoception signals are conveyed by dorsal root neurons that enter different laminae of the dorsal horn (Abraira and Ginty, 2013, Delmas et al., 2011, Todd, 2010). Since the result of hot plate test was not altered after dorsolateral penetration, the dorsal root might not be functionally injured. Instead, the dorsolateral penetration in this study might functionally affect part of neurons in the dorsal horn. The penetration might give stronger pressure to neurons responsible for light touch signals in the dorsal horn rather than those for nociceptive and thermoception signals. After the dorsal horn, fine/discriminative light touch signals are then transmitted to the brain via the dorsal columns. Since the dorsal column is located in the midline region, the midline penetration likely affected the transmission of light touch signals.

As for the transmission pathway after the dorsal horn, nociceptive and thermoception signals pass through the spinothalamic tract. Although the dorsolateral penetration in this study might give functional alterations in the dorsal side of the tract, performance in hot plate test was not altered in mice after dorsolateral penetration. Intact ventral side of the tract might be sufficient to convey nociceptive and thermoception information during hot plate test in mice.

Intriguingly, some literatures demonstrated crucial roles of interneurons located in the dorsal spinal cord in integration of tactile stimulation and corrective movements. RORα interneurons in the dorsal horn are involved in behavioral responsiveness to light touch but not responses to noxious and itch stimuli, and the RORα interneurons direct corrective reflex movements by integrating touch information with descending motor commands from the brain (Bourane et al., 2015). Thus, the dorsal horn neurons injured by the dorsolateral insertion might affect not only transmission of sensory information itself but integration of sensory signals and movements.

Likewise, responses to dynamic brush and static brush of the plantar surface of the hindpaws were changed in Zic2-neuron-ablated mice. Fine motor control was also impaired in mice lacking Zic2 spinal neurons. Accordingly, Zic2 dorsal column neurons proved to integrate sensitivity to light touch stimuli and corrective motor movements (Paixao et al., 2019). The midline penetration we employed might affect integration between light touch and movements.

The midline and dorsolateral penetration led to transient decrease in withdrawal response after light touch. Four weeks after the penetration, responsiveness to touch recovered to the level comparable to that before penetration. Transient decrease in the response after dynamic touch was also recognized in mice given contusion SCI (Kakuta et al., 2019). It is plausible to predict that a compensation regarding response to light touch occurred in the pathway responsible for transmission of sense of light touch. Consistently, compensation of the synaptic terminals and innervation was found after SCI in neonatal mice (Chawla et al., 2017). Even in the adult, collateral sprouting from preserved regions to the injury site was seen in the spinal cord (Fouad et al., 2001, Murray and Goldberger, 1974, Weidner et al., 2001).

As for symptoms and prognosis of penetrating SCI in humans, motor and sensory disturbance below the first thoracic vertebral level were found in a patient who stabbed his anterior neck using scissors (Deguchi et al., 2022). Emergency surgery did not lead to the improvement in both motor and sensory disturbance seen in the patient (Deguchi et al., 2022). By contrast, improved motor function was observed after surgery in some cases. For example, right lower limb paresis and deficits in pain, temperature and touch sensations below L2 were seen in a patient with penetrating SCI with screwdriver (Meena et al., 2016). After surgery, the patient improved to 4/5 power at hip, knee and ankle although sensory deficit persisted (Meena et al., 2016). In addition, stab injury at T12 vertebral body and the spinal cord resulted in hypoesthesia below the L1 level and motor deficit (Dogan et al., 2008). After surgery, the motor deficit was completely recovered. However, the hypoesthesia persisted (Dogan et al., 2008). Furthermore, although a patient’s gait became normal nine months later in the patient with penetrating injury by an iron fence point, improvement in the sensory deficit was not observed even 1 year after surgery (Ye et al., 2010). In these cases, recovery from sensory deficits was not enough. The mouse models produced in the current study might be suitable to explore therapeutic approaches for impaired response to tactile stimulation after penetrating SCI. However, further studies using mice with penetration at different sites are needed to test non-discriminative crude touch because non-discriminative crude touch information is transmitted to different tract in the spinal cord.

Another challenge is to determine whether dynamic touch given in this study modulates pathway for pain. Affective touch activates a subgroup of C-tactile fibers (Bjornsdotter et al., 2010), which can modulate pain (Meijer et al., 2022). Dorsal horn inhibitory system might be involved in the modulation (Meijer et al., 2022). Next challenge is to test whether stroking of the paintbrush used in the touch tests stimulates C-tactile fibers and the modulation of pain is seen after penetrating SCI. Such approaches will clarify precise location of injury sites that lead to dysfunction of specific sensory modality and interference between multiple sensory modalities.

CRediT authorship contribution statement

Taketo Otani: Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation. Shunsuke Nakamura: Writing – review & editing, Methodology, Investigation, Conceptualization. Tokue Mieda: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. Kazuhiro Nakamura: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Compliance with ethical standards

There are no financial/personal interest or belief that could affect their objectivity.

Mouse experiments were approved by the Animal Resource Committees of Gunma University (Approval number, 25–009). We followed NIH guidelines of mouse experiments and made every effort to minimize the number and suffering of the mice.

Funding

This research was supported by JSPS KAKENHI (Grant Number: JP23K15641) to T.M.

Acknowledgment

We thank the staff at the Bioresource Center and Life Science Hub, Core Facility Management and Technical Collaboration Center, Gunma University, for technical assistance.

Contributor Information

Tokue Mieda, Email: tmieda@gunma-u.ac.jp.

Kazuhiro Nakamura, Email: knakamur@gunma-u.ac.jp.

Data availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

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