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
Abstract Background: Immunohistochemical analyses of anterior cruciate ligament (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. Key terms: anterior cruciate ligament, remnant preservation, immunohistochemistry, nerve growth factor