This study demonstrates the successful administration and retention of NT-3 loaded poloxamer-based hydrogel at the intrathecal space of SD rats after contusion SCI, without negatively impacting the rats’ hindlimb functions and general well-being. The direct delivery of NT-3 via the hydrogel did not show significant trends in functional recovery and tissue regeneration.
To confirm the retention of the hydrogel in the subdural space, fluorescently labelled poloxamer gel was administered into the intrathecal space and its presence confirmed for up to 7 days (Fig. 1). The fluorescently labelled hydrogel residues were exclusively observed on the dorsal side of the spinal cord, below the dura, predominantly near the catheter entry point and injection site. Interestingly, previous research by Gupta et al. 25 had demonstrated the presence of thermosensitive fluorescently labelled hydrogel in the spinal cord 2 h after injection using a similar experimental setup. However, Gupta et al. did not investigate later time points.
Throughout the six-week study in SCI rats, the rats remained healthy, showing normal eating and drinking behaviour. Although there was some weight loss observed, it remained below 20% for all animals at any point, aligning with expectation for SD rats with contusion SCI 38,39.
To assess the rats’ functional recovery following direct delivery of NT-3 to the spinal cord, a BBB score test, horizontal error ladder test, and Von Frey testing was used. However, none of these tests revealed any significant trends in functional recovery. The variation in results across all groups, particularly observed in the BBB score results, where the maximum standard error of mean (SEM) ranged from 0.580 to 1.188, may have contributed to this lack of significance.
Interestingly, recent pre-clinical SCI studies have established the potential of NT-3 to enhance neural fiber density at the lesion site and promote functional recovery 21,31. For example, a study, which employed a different hydrogel-based drug delivery system and NT-3 demonstrated a significant increase in BBB scores between the treatment and control groups at week three 21. However, the BBB scores in that study were higher at day 28 compared to the BBB scores reported in our study, suggesting potentially increased injury severity in our experimental model 21. Similarly, in a 12-week study involving NT-3 and a different hydrogel-based system, no statistical differences between the control and treatment group were identified in BBB scores at any time point 22. This study, however, reported lower BBB scores at week 12 compared to the BBB scores reported in this study at week 6, indicating a more severe injury in their model compared to ours. Furthermore, it is worth noting that statistically significant improvements in BBB scores are not commonly observed with GF-based hydrogel treatments alone, especially at week 6 post-injury 40,41. Therefore, our research findings align with outcomes from previous studies.
For the horizontal error ladder test, we hypothesised a reduction in complete errors over the six-week period, coupled with an increase in hindlimb steps with no errors for the hydrogel + NT-3 treatment group. Despite stable or decreasing trends in the hydrogel and saline group, a slight upward trend in the percentage of complete errors was noticed for the hydrogel + NT-3 and NT-3 solution group (Fig. 3a), although none reached statistical significance. This lack of observed differences may be attributed to variations in the rats' speeds during their traversal of the 1 m error ladder, influencing both the number of steps taken and the errors made 42. For example, a slower run speed was generally associated with more stepping and an elevated risk of errors. Additionally, not all rats were able to immediately undertake the error ladder task, as determined by a cutoff BBB score being equal to or exceeding 10. This was particularly evident in the NT-3 solution group and hydrogel + NT-3 group, where two animals completed assessments only at the pre-injury and week 6 time points due to their BBB scores. However, contrary to the expectation that more practice/experience would enhance performance in the horizontal error ladder task, existing research has demonstrated that increased practice does not necessarily improve task proficiency 43. Moreover, results from a different study delivering NT-3 through gene therapy indicated a decrease in complete errors only after week 4, with statistical significance achieved only after 10 weeks, implying that our chosen time points may be too early to observe significant changes in this task 32.
The decrease in coordination post-injury may be caused by the exploratory nature of front limb placement, characterized by frequent repositioning on one rung before committing to another 42. This behaviour leads to the frequent uncoupling of fore and hindlimbs, suggesting the presence of tactile sampling of the terrain before committing to a specific stance strategy. Additionally, when assessing the coordination using the BBB data, more coordination was seen with hydrogel and hydrogel + NT-3, although not statistically significant. A similar trend was observed for NT-3 in a previous study 21.
Von Frey testing is a commonly used tactile sensory test to assess allodynia, a phenomenon frequently observed after SCI due to sensory system impairment 34,44. To ascertain the presence of allodynia or hypersensitivity, a threshold must be established. In this experiment, baseline values (Fig. 4b “Pre”) were defined as the allodynia threshold, as these thresholds varied across groups from around 20.7 ± 2.7 g for the hydrogel group to 24.5 ± 3.1 g for the saline group. Hypersensitivity/allodynia was assumed if a lower force elicited a response, while a non-allodynic responses were assumed if higher forces compared to baseline elicited a response. As seen in Fig. 4c, only the hydrogel group exhibited a non-allodynic response at week 1, and a near non-allodynic response at week 5. Moreover, at week 6 the hydrogel and hydrogel + NT-3 groups showed responses closer to non-allodynia at -1.3 ± 3.5 and − 1.9 ± 2.6, respectively, compared to -2.9 ± 2.0 and 3.1 ± 3.0 for the NT-3 solution and saline groups, respectively. This suggests a reduced degree of hypersensitivity/allodynia in the hydrogel and hydrogel + NT-3 groups, as an increase in force applied until paw withdrawal is associated with decreased hypersensitivity/allodynia 34. However, these differences at week 6 were not statistically significant. An alternative interpretation of this data is that the injury initially affects the rats' sensory ability in the hind paw, which is subsequently regained over time. As the rats recover motor function, evidenced by the increasing BBB scores, their sensory function also improves, resulting in a quicker response to the filament. This would explain the lower scores observed in all groups at week 6 (Fig. 4).
The subsequent analysis on tissue regeneration also did not reveal significant effects in neuronal regeneration, as assessed via β-tub 35 or GAP43 to β-tub-positive area ratio staining 7, 5-HT staining 22,26, or neuroinflammation and astrogliosis, as observed via Iba1 36 or GFAP 31 staining.
Although our study did not reveal observable effects in the β-tub-positive stained area, previous studies investigating hydrogel-delivered NT-3 for spinal cord injury in Long Evans rats demonstrated more extensive neural fibers sprouting at the injury site after 12 weeks compared to the control 22,31. It is important to note that the lack of a significant difference in our study might be attributed to a smaller sample size compared to these other studies, as well as the observed variability in our results. For example, a non-significant trend was apparent, indicating a larger β-tub-positive stained area in the hydrogel and hydrogel + NT-3 groups compared to the saline control group. However, it's important to acknowledge that the observed variability might mask potential significance.
The lack of observed significant effect on the GAP43 to β-tub-positive area ratio is also of interest, especially when compared to studies involving GFs delivered by hydrogel-based drug delivery systems like glial cell line-derived neurotrophic factor (GDNF) and acidic fibroblast growth factor (aFGF) 7,29,45. In those studies, an increase in GAP43-positive area in spinal cord injured SD or Long Eans rats indicated enhanced neuroplasticity. The observed contrast in results between our study and previous research could potentially be linked to the specific choice of growth factor. Furthermore, a general non-significant trend suggested increased neuroplasticity in the hydrogel and hydrogel + NT-3 groups compared to the control (saline) and NT-3 solution groups (Fig. 6). However, similar to the neuron levels across treatment groups, large variability in the hydrogel and hydrogel + NT-3 group might have masked potential significant effects.
Immunostaining with 5-HT antibodies was used to provide insights into the presence of serotonergic axons in the raphespinal tract 22,26. These axons originate from the nucleus raphe magnus and extend nerve connections to the dorsal and, to some extent, the ventral gray matter 46,47. Serotonergic neurons, identified by their 5-HT positivity, are distributed throughout every segment of the grey matter 26,46 (Fig. 7a) and play roles in nociceptive modulation and, to some extent, motor function control 46. Given the descending nature and high regenerative capacity of serotonergic axons, we conducted a separate analysis of both rostral and caudal sections 26. While we expected similar concentrations in rostral sections of 5-HT positive neurons among groups, we anticipated lower concentrations in caudal sections below the injury due to axon degeneration from descending neurons 20. Additionally, we expected increased sprouting of 5-HT positive neurons into the lesion area and higher concentrations in the rostral section for our hydrogel + NT-3 group, as it had been seen in other studies with hydrogel-delivered NT-3 in rats after SCI 22. However, no significant difference across the groups was detected.
In other studies, an increased number of serotonergic axons in the caudal section have been attributed to spared axons 26 and low levels in the caudal section have been linked to a lack of substantial hindlimb functional improvement 20. This aligns with our findings, as the ratio for 5-HT neurons did not show significant differences across the groups, and there was no significant difference across the groups in functional recovery. However, it's important to note that serotonergic axons are primarily associated with nociceptive control 46. Interestingly, our von Frey data aligns with the trends in 5-HT ratios, as the hydrogel group exhibited the lowest ratio in 5-HT positive neurons, suggesting fewer nociceptive connections, correlating with the reduced allodynia observed with von Frey testing. However, it is important to note that these trends were not significant.
No significant differences were also noted in the Iba1-positive area across the treatment (Fig. 8b). It is noteworthy that microglial activation can play a beneficial role in neural repair by promoting the release of anti-inflammatory growth factors and cytokines 48. Additionally, another study also reported a lack of reduction in the inflammatory response in the spinal cord following hydrogel-delivered growth factor treatment 26, suggesting that the absence of a difference might not necessarily have a negative impact.
GFAP staining was used to detect astrocytes and to indicate glial scar formation 31. Interestingly, no significant difference in GFAP-positive area was found across the treatment groups (Fig. 8d). We initially anticipated a reduction in the GFAP-positive stained area, as another study had reported this following hydrogel-delivered NT-3, hypothesized to be due to NT-3 encouraging cell infiltration 31. However, a different study showed a similar outcome in a hydrogel-based drug delivery system for NT-3, aligning with our study, where no significant difference in GFAP staining around the lesion site was found 21. This suggests that GFAP reduction might not always be directly related to improved outcomes.
To ensure the translatability of the results in this study, we used the most clinically relevant contusion injury model of SCI, in which a small blunt force impact is delivered to the cord 16,49. Notably, only a few of the other pre-clinical hydrogel studies used a contusion injury model of SCI (5 out of 30 studies) 26,50. Instead, the majority of these studies have employed a transection injury model (11 out of 30 studies) or compression injury model (14 out of 30 studies) 16,49. Transection models mimic rare laceration injuries instead of the more common compression and most common contusion injuries globally 16,51.
Furthermore, in this study, a hydrogel volume of 10 µL was selected to minimise its impact on the cerebral spinal fluid (CSF) flow. Although there is no rodent-specific information available, previous studies have indicated that intrathecal injections at volumes ranging from 33 to 42% of total CSF volume are tolerable in humans and non-human primates 52. Considering that the CSF volume in rats is around 150 µL 53, the chosen 10 µL volume accounts for less than 10% of the total CSF volume, making it well below the threshold of concern. This aligns with our observations as the fluorescently labelled gel did not appear to obstruct the CSF flow, spreading along the dorsal area of the spinal cord. Furthermore, there were no signs of locomotor problems or instability in uninured rats, which suggest that there was no damage to the spinal cord. Additionally, while there was weight loss observed in our pilot study with injured animals, the weight loss was less than 10% throughout the seven days, which is consistent with what has been observed in previous studies 38, suggesting no further damage from the hydrogel injection.
Overall, in this study a lack of statistical significance was detected. This absence of statistical significance could be attributed to multiple factors, with the timing of the treatment intervention being a potential aspect. A recent study by Squair et al. 54, for example, emphasised the need for a more comprehensive understanding of the specific temporal and spatial requirements in delivering GFs to achieve functional recovery in SCI. In their study, a complex regeneration strategy was used, involving viral GF overexpression, temporally delayed delivery of GFs, and the generation of a chemoattraction gradient through dorsally implanted biomaterial depots of GDNF. Their study demonstrated significant walking recovery after complete spinal cord injury, which highlights the importance of precise considerations regarding temporal and spatial requirements for achieving functional recovery. However, further systematic investigations into the exact requirements might be warranted.
Another potential factor revolves around the uncertainty regarding the optimal in vivo dosage of NT-3 needed for functional recovery 21,31. While there is an increasing focus on determining NT-3 concentrations in in vitro cell-based assays 6, relatively few studies have explored the optimal NT-3 concentration in vivo 22. Determining the optimal NT-3 concentration is further complicated by varying results in in vitro studies, suggesting concentrations ranging from 100 ng.mL-1 31 to 200 ng.mL-1 in the presence of inhibitory chondroitin sulfate proteoglycans 6 for achieving statistically significant neurite outgrowth. In in vivo rat SCI models, studies have used NT-3 doses ranging from 20 ng 31 to 2 µg 21. Notably, using the same amount of NT-3 as in our study (2 µg) in a similar injury model demonstrated significant effects on axon growth and some functional recovery 21. However, in this specific study, a higher total cumulative amount of NT-3 (1.72 µg) was detected in vitro, compared to approximately 1.14 µg of bioactive NT-3 in our study (extrapolated from in vitro release data). Furthermore, the study explored the amount of NT-3 in the spine at different depths and time points, which are details that have not been explored in our study. Future work could, therefore, focus on further validation of the delivery system to determine how much NT-3 is released in vivo and its distribution. This also emphasises the critical importance of ensuring that the effective concentration reaches the target site. Additionally, considering the potential need for repeated doses of NT-3 for functional regeneration, our system released relatively larger amounts of NT-3 for 14 days, with lesser amounts being released from 14 to 28 days. More systematic studies are, therefore, needed to determine the optimal GF concentrations in vivo and how to release them optimally using drug delivery systems.
We also note that our study was only six weeks in duration, whereas other studies extended up to 12 weeks 26,41,55. However, in these longer-duration studies, improvements resulting from the treatment were observable from three to four weeks onwards 26,55. Therefore, we expected significant improvement in functional recovery and tissue regeneration to be evident by week six 21. Nevertheless, it is possible, especially for treatments that require longer release profiles or multiple doses, that a more extended study duration would reveal evidence of treatment efficacy. Additionally, it's worth noting that statistically significant improvements in functional recovery are not consistently observed 40. Potential variability in injuries, as reflected by variance in most in vivo results, could also contribute to the observed lack of statistical significance. This aligns with a widely acknowledged issue of reproducibility of SCI studies 56 and highlights the importance of considering the heterogeneity of the injury including factors such as lesion size, severity, and location 16. For example, current research often uses the lesion size as marker for injury severity. However, the neuroanatomical-functional paradox highlights the significant impact of the SCI site's position compared to other factors, potentially giving rise to reproducibility issues 57. Therefore, slight variations in injury positions may have affected injury severity and study outcomes.
Moreover, our study could have benefited from including more animals in the tissue response analysis, similar to other studies with larger sample sizes ranging from 3 to 8 animals, and a smaller region of interest, such as 250 µm into the tissue on all sides of the lesion 26,58. However, the inclusion of more animals could potentially increase variance in results, as our selection criteria aimed to resemble the closest mean of the BBB score. Additionally, a larger region was chosen to ensure that the injury site was completely encompassed in all tissues.
Despite these challenges, we successfully ensured the delivery of the GF-loaded hydrogel directly to the cord without causing further damage. The site of injection requires careful consideration, and our study demonstrates that injecting into the intrathecal space between the dura mater and spinal cord is a preferable approach, as supported by other studies 21. This approach contrasts with directly injecting the hydrogel into the spinal cord at the injury site or placing the gel on top of the injury site on the damaged dura. Such direct methods could potentially cause damage or hinder the ability of growth factors to pass through the dura membrane 7,26,27, which was not observed in this study.