Quantitative assessment of neural elements in rat model using nerve growth factor after remnant-preserving anterior cruciate ligament reconstruction: a hematoxylin and eosin stain and immunohistochemical study

Background: Immunohistochemical analyses of (ACL) allografts following remnant-preserving ACL reconstructions using Achilles tendon allografts have provided evidence for the presence of neural elements. In this study, we aimed to examine the expression of neural elements and quantify the presence of neural cells in ACL remnants and Achilles allografts using nerve growth factor (NGF) therapy after remnant-preserving ACL reconstruction. Methods: Experiments were conducted on 5 pairs of rats (approximately 8 weeks old and weighting 320 g at the time of surgery). Longitudinally-split Achilles tendons from the paired rats were freshly frozen and later defrosted with warm saline and allografted onto the right ACL of the other, which was partially detached at the femoral attachment site. A sham operation was conducted on the left knee to be used as Control. NGF was injected in both the knee joints 1 week after surgery. The presence of neural cells in the ACL of the sham-operated knee, allografted Achilles tendon, and ACL remnant was examined 6 weeks post surgery using H and E and immunohistochemical staining. Results: H and E staining did not reveal neural cells in any of the three groups. However, immunohistochemical analysis showed the presence of nestin-positive neural elements in normal ACL as well as ACL remnants. Additionally, neural elements were examined in 7 of the 8 (87.5%) allograft tissues. Quantitative analysis showed no difference in the number and area of nuclei among the three groups. However, the number and area of neural cells in Achilles allograft were significantly lower than in the other two groups (p=0.000 and p=0.001, respectively). Conclusion: Our observations indicate that ACL remnants promote new ingrowth and persistence of neural cells. We suggest that the ingrowth of neural elements could support the persistence and new ingrowth of mechanoreceptors, thereby enhancing the functional stability of knee joints. Moreover, the expression of neural cells in Achilles allograft was lower than that of normal ACL or ACL remnants in the quantitative evaluation, thereby confirming the essential role of ACL remnants in knee joint functionalization.


BACKGROUND
The functional instability of a knee joint is majorly caused due to the lack of coordinated muscle stabilization, thereby inciting pertinent interest in unraveling methods to protect mechanoreceptors in the knee joints [1,2]. It has been reported that remnant-preserving ACL reconstruction technique preserves mechanoreceptors and renders positive results [1,3,4,5,6,7]. The current study aimed to determine whether the remnant preservation technique preserves the existing mechanoreceptors or leads to re-innervations.
Evidence has demonstrated the growth of mechanoreceptors in reconstructed ACLs.
For instance, Barrack et al. reported increasing of mechanoreceptors in ACL grafts in dogs 6 months after surgery compared to their normal patellar tendon [8]. Denti et al. reported that mechanoreceptors were present in the bone-patellar tendonautografts in the knees of sheep 3 months after surgery, and also in human knees with failed autograft at 9 and 10 years after surgery [9]. These findings suggested that ACL remnants are a possible source of re-innervation of the tendon graft.
However, previous histological studies failed to validate the presence of mechanoreceptors in ACL allografts [10]. In addition, few studies have quantitatively evaluated neural elements in ACL remnants and ACL allograft.
The nerve growth factor (NGF) is widely used clinically for its significant roles in supporting neuronal survival, peripheral nerve growth, nutritional adaptation, nerve regeneration and fracture repair [11]. However, it has not been delineated whether these receptors effect on re-innervation or proprioception. However, the it is important that the persistence of mechanoreceptors after remnant-preserving ACL reconstruction. Thus, stimulating the expression of neural cells using NGF, we tried to validate the superiority of remnant preservation technique.
The aim of present study was to investigate the effects of administering NGF following remnant preservation technique by assessing the presence of nerve cells immunohistochemically in the allograft and remnant ACL. In addition, neural elements in ACL remnants and ACL allografts were quantitatively evaluated using ImageJ particle analysis. We hypothesized that remnant tissues following ACL reconstruction as well as Achilles allografts would contain neural cells, which was identified using immunohistochemistry.

Animals
Experiments were conducted on 10 adult male rats (Rattus norvegicus albinus, Samtako®, Korea), approximately 8 weeks old, and weighing 320 g at the time of surgery. All the rats were numbered using ear tags. Rats were housed in accordance with the National Institutes of Health guidelines, kept in a vivarium, maintained at 20-25 °C and 60% humidity with 12 hours alternating light-dark cycles (7 am-7 pm), and provided food and water ad libitum.

Surgical procedure
The remnant-preserving ACL allograft Arthrotomy was performed after intraperitoneal (IP) administration of general anesthesia [a mixture of Zoletil (50 mg/kg, Virbac Laboratories, France) and Rompun (10 mg/kg, Bayer, Korea)]. In pairs, the Achilles tendon was obtained from a donor rat and used as an allograft for ACL reconstruction in the right knee of the recipient rat. Briefly, an approximately 1.5 cm longitudinal skin incision was made on the Achilles tendon insertion site in the left ankle. After longitudinally splitting the 1.5 cm sized Achilles tendon of each rat, it was fresh frozen at -80 °C for 5 minutes in a deep freezer. Then, the Achilles tendon was defrosted with 47 °C warm saline for 5 minutes (Fig. 1) and used as allograft.
For the ACL reconstruction, an approximately 1.5 cm incision was made on the right knee. Then, the joint was exposed by transposing the patella laterally after a parapatellar incision was made with the knee in flexion. The ACL was detached at femoral insertion, but tibial insertion was maintained. Subsequently, the Achilles tendon was allografted onto the right ACL which was partially detached at the femoral attachment site. Suture fixation was used on Femoral, tibial ACL anatomical attached site (Fig. 2). Irrigation was performed on the joints, and the capsule and skin were closed with interrupted sutures.

The Sham operation
A sham operation was conducted on the left knee as a control. The knee joints were exposed with the same incision. Also irrigation was performed on the joints, and the capsule and skin were closed with interrupted sutures. classified into four types according to prior study (type I, a spherical or ovoid Ruffini corpuscle; type II, a columnar concentric circular Pacini corpuscle; type III, a spindle-shaped Golgi corpuscle; and type IV, a non-myelinated free nerve ending) [12]. H and E stained sections were assessed for the presence of mechanoreceptors following the stated classification.

Immunohistochemical staining
Sliced tissues on the coverslips were washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 minutes, permeabilization with 0.1% Triton X-100 in PBS for 5 minutes at room temperature.
After washing three times with PBS, sections were blocked with 1% bovine serum albumin (BSA) for 1 hour at room temperature. Sections were incubated with antinestin antibody (Sigma, St. Louis, MO) (diluted in blocking solution (1% BSA in PBS)) for 1 hour at room temperature in a shading box. Subsequently, the tissue sections were washed three times with PBS and incubated with secondary antibody, Alexa Fluor 555-conjugated rabbit anti-goat antibody (Invitrogen, Grand Island, NY) for 1 hour at room temperature. Alexa Fluor 488-conjugated phalloidin (Invitrogen, Grand Island, NY) was used for F-actin staining. Nuclei were stained with 4,6-diamidino-2phenylindole (DAPI, Santa Cruz Biotechnologies, Santa Cruz, CA). Images were captured using confocal microscope (Olympus, FV-1 mm).

Quantification with particle analysis
After immunohistochemical staining, quantitative analysis was performed on each image using ImageJ (National Institutes of Health, Bethesda, MD). The number and area of nuclei that were stained with DAPI and neural cells that were stained with anti-nestin antibody were calculated. Several attached cells in each image were optically separated using a watershed separation tool provided by the ImageJ software. Following this, the area and number of cells were quantified using ImageJ particle analysis.

Statistical Analysis
Statistical analysis was conducted using SPSS for Windows, version 12.0. Kruskal Wallis test was used analyze the immunohistochemical data (with the 95% confidence level). P-values < 0.05 were considered significant. Where indicated, Mann-Whitney post-hoc analysis was performed after the Kruskal-Wallis test, with a significance level set at p < 0.16. The power of group comparison was analyzed using G*Power 3.1.9.2, where this study achieved a power of 0.68 for detecting differences with α = 0.05.

RESULTS
Two of 10 rats expired during the experiment (at 2 weeks and 4 weeks postsurgery). The experiment was completed with the remaining 8 rats.
H and E examination confirmed ligamentous ACL tissues and synovium in all the sections (Fig. 3). However, neural cells or mechanoreceptors could not be detected in normal ACL, ACL remnants, and Achilles allograft tissues through H and E staining (Fig. 3).
In the immunohistochemical study, nestin was expressed in all normal ACL tissues that were injected with NGF. In addition, nestin was expressed in all ACL remnants and in 7 of 8 (87.5%) allograft tissues that were processed with NGF (Table 1).
Nestin-positive cells suggested the possible presence of neural cells in the ACL remnant tissue and Achilles allograft (Fig. 4). We did not observe any significant difference in neural cell expression among the normal ACL, ACL remnants, and Achilles allografts with the immunohistochemical study (p = 0.368). Quantitative analysis showed no difference in the number and area of nuclei among normal ACL, ACL remnants, and Achilles allografts. However, the number and area of neural cells were significantly different among the groups. Post-hoc analysis revealed that the number and area of neural cells in Achilles allografts was smaller than that of normal and remnant ACLs (p = 0.000 and p = 0.001, respectively).

DISCUSSION
This study used immunohistochemical analysis to shed light on a critical aspect of the localization of neural elements. We provide evidence that the neural elements are found in not only remnant ACL but Achilles allografts. In the quantitative test, neural cells of ACL remnants were not found to be different from those of normal ACL. These results indirectly demonstrate that ACL reconstruction with remnant preservation could better preserve proprioception and be a resource for reinnervation in reconstructed allografts.
Proprioception is a key factor in maintaining the stability of knee joint against functional deficiencies after ACL reconstruction. Many mechanoreceptors known to be distributed in ACL and remnant tissues contribute to the proprioceptive function of the knee [13,14]. Some previous studies reported the presence of mechanoreceptors in ACL remnants after ACL reconstruction with remnant preservation technique [5,15,16,17]. Previous study reported the presence of mechanoreceptors in ACL remnants 3 years after ACL rupture [17]. In another recent study, normal proprioceptive fibers and mechanoreceptors were reported in 50% of remnant ACL using neurofilament protein staining [16]. They also showed the difference between mechanoreceptors in healthy and injured ACLs and highlighted their clinical significance [15]. Lee et al. reported the presence of mechanoreceptors in about one-third of the ACL remnants, which was less than expected [5]. In this study, neural cells were identified in ACL remnants, which was not different from normal ACL upon quantitative evaluation. This outcome suggests that remnant-preserving ACL reconstruction could preserve proprioception and have a positive effect on knee joint functions.
Conversely, few studies suggest the possibility that ACL remnants could serve as a However, in their study, neural elements were not observed in Achilles allograft tissues [20]. Therefore, we investigated the expression of mechanoreceptors in remnant tissues and allografts following remnant-preserving ACL reconstruction using NGF therapy. In the present animal study, the presence of neural elements was confirmed in remnant tissues and allografts using immunohistochemical methods with NGF, a well-known growth factor for nerve cell proliferation.
The presence of neural cells in Achilles allografts supports the potential regeneration of neural elements during remnant-preserving ACL reconstruction. This was, however, not supported in previous studies [10,19].
Many studies have evaluated proprioception, as well as the function of mechanoreceptors in reconstructed ACLs [5,10,13,14,15,16,17]. However, it is difficult to compare outcomes between these studies because of variations in the experimental methods and other external factors. Recently, immunofluorescence and immunohistochemistry using specific antigen-antibody reactions have been used to detect nerve fibers, producing more reliable and relevant results [5]. In this study, we visualized mechanoreceptor-positive cells using immunohistochemical assessment with monoclonal antibodies against nestin. To identify the presence of nestin-positive cells in the remnant tissues, the cells were treated in vivo with NGF which was an important factor in the growth and maintenance of sensory and sympathetic neurons. Previous studies reported that NGF application could be used to promote the healing of ACLs [21]. They proposed that NGF promoted reinnervation and angiogenesis in healing rat ACLs. Therefore, we evaluated the expression of mechanoreceptors in remnant tissues and allografts after remnantpreserving ACL reconstruction using in vivo administration of NGF in rats. The results of the current study indicate that NGF promotes re-innervation of Achilles allografts.
Few studies have attempted to quantify the proprioceptive potential of the injured ACL stump [9,16,17]. However, none of these studies have quantified their immunohistochemical findings. We analyzed the number, area of nuclei and neural cells that were each stained with DAPI and anti-nestin antibody. The expression of the quantitative evaluation, although nerve growth was promoted by NGF.
Therefore, to investigate the clinical significance of the expression of small amounts of neural elements in allografts, further research on the differences in proprioceptive function after remnant-preserving ACL reconstruction using allografts will be required.

CONCLUSION
The results from this study demonstrate that ACL remnants promote the new ingrowth and persistence of neural cells. We suggest that the ingrowth of neural elements could support persistence and new ingrowth of mechanoreceptors and functional stability of the knee joints. However, the expression of neural cells in Committee of Wonkwang University (WKU17-108). The procedures for animal experiments were in accordance with requirements in Declaration of Helsinki.

Figure 1
Achilles tendon was grafted for ACL reconstruction. A: Achilles tendon exposure, B, C: split A Figure 2 Achilles tendon was allografted to each other's left ACL. A: ACL exposed, B: ACL is partially d Figure 3 . In all cases, no mechanoreceptors were present on H&E staining. A: normal ACL, (control sp Figure 4 Immunohistochemical findings of nerve cells. Nuclei were stained with 4,6-diamidino-2-pheny Several attached cells in each image were separated using the watershed separation tool ba