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ORIGINAL ARTICLE
Year : 2022 | Volume
: 21
| Issue : 4 | Page : 361-365
The efficacy of new neuronal growth factor in the healing of the sciatic nerves in rabbits
Mir Sadat-Ali1, Dakheel Abdullah Aldakheel1, Majed N Alabdali2, Dana T Aljaafari2, Abdulla A Alsulaiman2, Abdallah S Alomran1, Ayesha Ahmed3, Fahd A Alkhamis2
1 Department of Orthopaedic Surgery, King Fahd Hospital of the University, Imam Abdul Rahman Bin Faisal University, Alkhobar, Saudi Arabia
2 Department of Neurology, King Fahd Hospital of the University, Imam Abdul Rahman Bin Faisal University, Alkhobar, Saudi Arabia
3 Department of Pathology, King Fahd Hospital of the University, Imam Abdul Rahman Bin Faisal University, Alkhobar, Saudi Arabia
Correspondence Address:
Mir Sadat-Ali
Department of Orthopaedic Surgery, King Fahd Hospital of the University, Imam Abdul Rahman Bin Faisal University, AlKhobar 31952
Saudi Arabia
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_84_21
Abstract
Background and Objective: Regeneration of nervous tissue is unpredictable and an ideal growth factor to influence the healing of the injured nerves is not available. A recent study in rats had shown that a new neuronal growth factor (NNGF) was effective in the early healing of the sciatic nerves. The aim of this experimental study is to test the efficacy of NNGF in the healing of iatrogenic division of the sciatic nerves in a larger animal (rabbits). Methods: White New Zealand 20 male rabbits of 6 months of age were divided into two groups. Intramuscular ketamine and xylazine were used to anesthetize the animals. The sciatic nerves were divided using scalpel blade 15 and 10/0 Vicryl was used to repair the divided neural tissue. In the study group, 10 mg/kg body weight of NNGF was instilled on the top of the divided nerves and the wound was closed. At 4 weeks, the operated limbs were observed for any trophic skin changes. Nerve conduction studies were carried out using train-of-four-Watch SX, Organon (Ireland) Ltd., and Ireland. The rabbits were put to death humanely and the sciatic nerves were removed and delivered to the pathologist in 2% formalin. The pathologists were blinded about the two groups. Results: Electromyographic study done at 4 weeks showed in the untreated group; the mean twitches 1-T4 was 0.45 ± 0.31% and in the treated group, the average was 77.912 ± 5% (P > 0.001). Microscopic anatomy in the treated group revealed prominent healing by regeneration was evidenced by showing growth of its proximal segments into an empty endoneurial tube which was not seen in the control group. In the control group, the nerves showed no histological element of healing by regeneration. Conclusions: NNGF proves that in a larger animal at 4 weeks profoundly influenced early regeneration of experimentally created divisions of myelinated nerve tissue.
Abstract in French Résumé
Contexte et objectif: La régénération des tissus nerveux est imprévisible et un facteur de croissance idéal pour influencer la guérison des nerfs blessés n'est pas disponible. Une étude récente chez le rat a montré qu'un nouveau facteur de croissance neuronal (NNGF) était efficace dans la guérison précoce des nerfs sciatiques. L'objectif de cette étude expérimentale est de tester l'efficacité du NNGF dans la guérison de la division iatrogène des nerfs sciatiques chez un animal plus grand (lapin). Méthodes: White New Zealand 20 lapins mâles de 6 mois ont été divisés en deux groupes. La kétamine intramusculaire et la xylazine ont été utilisées pour anesthésier les animaux. Les nerfs sciatiques ont été divisés en utilisant la lame scalpel 15 et 10/0 vicryl ont été utilisés pour réparer le tissu neural divisé. Dans le groupe d'étude, 10 mg / kg de poids corporel du NNGF ont été inculqués sur le dessus des nerfs divisés et la plaie a été fermée. À 4 semaines, les membres opérés ont été observés pour tout changement de peau trophique. Des études de conduction nerveuse ont été menées à l'aide de SX Train-of-Nat-Watch, Organon (Ireland) Ltd. et Ireland. Les lapins ont été mis à mort avec humanité et les nerfs sciatiques ont été retirés et livrés au pathologiste dans 2% de formol. Les pathologistes ont été aveuglés sur les deux groupes. Résultats: électromyographique L'étude réalisée à 4 semaines a montré dans le groupe non traité; Les contractions moyennes 1 à T4 étaient de 0,45 ± 0,31% et dans le groupe traité, la moyenne était de 77,912 ± 5% (p> 0,001). L'anatomie microscopique dans le groupe traité a révélé une guérison importante par régénération a été mise en évidence en montrant la croissance de ses segments proximaux dans un tube endoneurial vide qui n'a pas été observé dans le groupe témoin. Dans le groupe témoin, les nerfs n'ont montré aucun élément histologique de guérison par régénération. Conclusions: NNGF prouve que chez un animal plus grand à 4 semaines, a profondément influencé la régénération précoce des divisions créées expérimentalement du tissu nerveux myélinisé.
Mots-clés: Facteurs de croissance, régénération nerveuse, nerfs sciatiques
Keywords: Growth factors, nerve regeneration, sciatic nerves
Introduction 
Even though the clear elucidation of nerves and nervous tissue was done in the 10th century.[1],[2],[3],[4] It took centuries before the function of the peripheral nerves and their healing could be clearly understood.[5],[6] Injuries to the peripheral nervous system particularly to the upper limbs are common and cause extensive disability, distress, and morbidity.[7] As per the classification of nerve injuries by Sunderland[8] Class V or Seddons Neurotmesis,[9] the recovery is unpredictable to none and these injuries occur due to trauma with a prevalence of 1.3%–2.8%[10],[11] costing roughly $150 billion yearly in the United States.[12]
Microsurgical advancements in microsurgical and reconstructive techniques have improved the outcome of peripheral nerve injuries but a complete recovery is usually infrequent and associated with long-term morbidity. Recently, growth factors have come in vogue in the healing of bone, nerves, and soft tissues. Growth factors are normally ether polypeptides or steroid hormones that influence cellular activity at the molecular level helping cellular growth, proliferation, differentiation, and maturation.[13] Growth factors related to the members of the neurotrophin family have shown a promising effect on the healing of the peripheral nerves.[14],[15] Recently, Zhang et al.[15] showed that different growth factors are expressed, during peripheral nerve regeneration.
Earlier new neuronal growth factor (NNGF) was used in rats healing of the sciatic nerves and showed early healing of the divided sciatic nerves[16] To further develop the NNGF, we conducted this study in rabbits.
Methods The study was approved by the Institutional Review Board (IRB) of the Imam AbdulRahman Bin Faisal University, Dammam, Saudi Arabia. The isolation and production of NNGF are described elsewhere.[16] Twenty white New Zealand male rabbits of 6 months of age were divided into two groups of 10 each. The rabbits in both the groups were comparable for the age, weight and had the same nutritional status as they were bred in house. Animals were anesthetized using a standard dose of ketamine mixed with xylazine. The eyes of the animals were protected to avoid corneal injuries. The operated area over the right hind limb was cleaned and scrubbed using 2% betadine. The sciatic nerves of the right hind limb were isolated. The sciatic nerves were divided with scalpel blade 15 and sutured using 10/0 Vicryl. In the study group, 10 mg/kg body weight of NNGF was instilled at the site of the division of the nerves and the wound was closed. In control animals, the wound was irrigated and closed. All animals received intramuscular Zinacef at a dose of 30 mg/kg body weight and for analgesia diclofenac 5 mg/kg body weight for pain. The animals were kept in individual cages with unrestricted water and a standard pellet diet. At the end of 4 weeks, the distal limbs were observed for skin changes. Nerve conduction studies were carried out using train-of-four (TOF)-Watch SX, Organon (Ireland) Ltd., Ireland. The rabbits were put to death humanely after anesthetizing using an intramuscular injection of a combination of xylazine 5 mg/kg and ketamine 35 mg/kg of body weight and once the animals were fully anesthetized, decapitation was performed, then the sciatic nerves were removed and delivered to the pathologist in 2% formalin. The pathologists were blinded about the two groups. The study was monitored by the monitoring office for research and research ethics of the university, as instituted by the Ministry of Higher Education, Kingdom of Saudi Arabia. Data were analyzed using the SPSS Inc Version 21, Chicago, USA. Data were expressed as mean ± standard deviation. Statistically significant differences between the different groups were determined with the Student's t-test using a P < 0.05, which is considered to be statistically significant with confidence interval of 95%.
Results All the animals survived the surgery and the follow-up period. After 4 weeks of the division of the sciatic nerves, extensive skin ulceration was observed on the distal foot of the control group [Figure 1] compared to the study group [Figure 2]. Electromyographic studies postrepair showed in the untreated animals, the average twitches 1-T4 was 0.45 ± 0.31% and in the treated group, the average was 77.912 ± 5% (P = 0.0001). The details of the TOF-Watch recordings of the treated and untreated groups are given in [Table 1] and [Table 2]. [Table 3] shows the findings between the untreated and the treated groups with P < 0.0001.
Figure 1: Extensive ulceration of the operated site after 4 weeks in the control group
Figure 2: Minimal ulceration of the operated site after 4 weeks in the study group
Table 3: Comparison between Electromyography finding between the control and the study Group of RabbitsIn the treated group, the strength of the twitches, which reflected the evoked response, were comparatively better distal to the repair as compared to the untreated group indicating robust healing. The strength of the twitches indicated that there was no conduction block in the treated group.
[Figure 3]a,[Figure 3]b,[Figure 3]c,[Figure 3]d shows the lower magnification microphotograph with arrows pointed at the sprouting axons. Prominent nerve healing by regeneration is represented by the growth of the proximal segment of the nerve into an empty endoneurial tube. [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d shows histopathological pictures of control animals showing collagen deposition and fibrosis with no element of nerve healing by regeneration. [Figure 4]c shows trichrome stain highlighting the collagen in blue color in the nerve tissue, ×10 high pass filter (HPF) 4 D also shows trichrome stain highlighting the collagen in blue color in the nerve tissue, ×40 HPF. [Figure 5]a,[Figure 5]b,[Figure 5]c,[Figure 5]d shows the growth of axons in endoneurial tubes at the site of injury, whereas [Figure 6]a,[Figure 6]b,[Figure 6]c,[Figure 6]d of the control group showing extensive calcification and variable inflammation, representing degenerative changes. [Figure 6]a and [Figure 6]b shows Calcification (single arrow) and chronic inflammation with prominent eosinophils (double arrow).
Figure 3: (a-d) Study Group 1: prominent nerve healing by regeneration represented by the growth of the proximal segment of the nerve into an empty endoneurial tube. (a) H and E × 10HPF. (b) H and E × 20HPF. (c) H and E × 20HPF. (d) H and E × 40HPF. HPF: High-pass filter
Figure 4: (a-d) Control Group 2: Collagen deposition and fibrosis in a nerve with no element of nerve healing by regeneration. (a) Nerve tissue H and E × 10 HPF. (b) Nerve tissue H and E × 40 HPF. (c) Trichrome stain highlighting the collagen in blue color in the nerve tissue, ×10 HPF. (d) Trichrome stain highlighting the collagen in blue color in the nerve tissue, ×40 HPF. HPF: High-pass filter
Figure 5: Study Group: Growth of axons in endoneurial tubes at the site of injury. (a) Growing axons in endoneurial tubes highlighted by arrows, H and E × 10HPF. (b) Growing axons in endoneurial tubes highlighted by arrow, H and E × 20HPF. (c) Vacuolar degeneration seen in the growing axons highlighted by arrows, H and E × 40HPF. (d) Margin of sprouting axon in the endoneurial tubes at the site of injury, H and E × 10HPF. HPF: High-pass filter
Figure 6: Control Group: Extensive calcification and variable inflammation, foreign body-type giant cell reaction representing degenerative changes. (a) Calcification (single arrow) and chronic inflammation (double arrow), H and E × 10. (b) Calcification (single arrow) and chronic inflammation with prominent eosinophils (double arrow), H and E × 40. (c) Foreign body giant cell reaction in an inflammatory background. The arrow points toward the foreign body type giant cells. H and E × 40. (d) Foreign body giant cell reaction around an eosinophilic material, in an inflammatory background. The arrow points toward the eosinophilic material. H and E × 40 Discussion This study shows that NNGF positively influenced the recovery of the iatrogenic sectioned sciatic nerves in rabbits. The two-point assessment electromyographic and histologically both show significant differences between the treated and untreated groups of animals. In the initial study of NNGF in rats,[16] the sciatic nerves were tested after 8 weeks and in the study, we used a larger animal and reduced the duration of tests from 8 weeks to 4 weeks, but the results were similar.
When the peripheral nerve gets injured, Schwann cells start the process of the regeneration process,[17] but in the healing process, many growth factors are expressed in the injured nerve, which further increases cellular activity for repair of the injured neural structures.[18]
Recently, studies have shown convincingly the efficacy of growth factors in nerve healing.[19] Various growth factors have been in the trials, which influence different stages of nerve healing. Fibroblast growth factors which were first identified as a factor which was released from the injured nerve supposed to play a major role in the regeneration injured nerve.[20] Growth factors such as nerve growth factor (NGF) and[21] vascular endothelial growth factor (VEGF) which have an effect on neovascularization; were reported that VEGF has positive effects on injured nerves.[22] Even though NGF, a neurotrophic factor which was discovered in the early 1950s showed that peripherally innervated tissues produced and released NGF which helps in the functional integrity of the peripheral nerves, exogenous NGF in human trials was limited due to its side effects.[23]
Zhang et al.[15] studied injured rat sciatic nerve stumps at 0 h, 1 day, 4 days, 7 days, and 14 days and found different growth factors were expressed for axon growth and nerve regeneration. This suggests that growth factors either endogenous or exogenous are essential in the healing of neural tissues. Serrano et al.[24] found the normal NGF level in the blood to be 194 ± 25 pg/ml which is required for the normal maintenance of the regeneration of the nervous tissue. Injury probably demands much more for normal healing to occur and with the lack of an endogenous growth factor, an exogenous growth factor can stimulate the healing.
The limitation of this study was that we did not perform further analysis using electron microscopy which would have shed more light on axonal count and depth of the healing. The strength is that NNGF has shown its pharmacological potential even in a larger animal with no complications whatsoever.
Conclusions This study has demonstrated that under influence of NNGF, experimentally divided sciatic nerves heal better and faster than the control group. We believe that there is ample potential in NNGF in inducing neural tissue healing. Further assessment by way of toxicology studies and phase I human trials are warranted.
Ethics approval
Ethical approval for the study was obtained from the IRB of Imam AbdulRahman Bin Faisal University, Dammam. The animal study was performed as per the outlined fundamental principles and as per the requirement of the International Council for Laboratory Animal Science.
Consent for publication
All authors give their consent for publication. Availability of data and material: The data are available at https://data.mendeley.com/[email protected]
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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