Ultrasound-guided injections of amniotic membrane/umbilical cord particulate for painful neuropathy of the lower extremity

Abstract Treatment of peripheral neuropathy remains a challenge. It has been shown clinically that cryopreserved human amniotic membrane (AM) and umbilical cord (UC) reduce pain, and they may serve as a beneficial treatment option for peripheral neuropathy. Here, we report findings from a single-center, retrospective review of peripheral neuropathy patients treated with AM/UC particulate. Seventeen patients with recalcitrant diabetic (n = 8), idiopathic (n = 7), or chemotherapy-induced peripheral neuropathy (n = 2) were included in the study. At presentation, all 17 patients complained of pain, along with numbness (n = 10), paresthesia (n = 8), poor balance (n = 9), poor range of motion (n = 5), or weakness (n = 7). After an average of 2.7 injections of AM/UC particulate per extremity, symptoms improved by 30.0 ± 24.5% at 1 week, 46.6 ± 29.9% at 1 month (P < .005), 70.7 ± 14.3% at 2 months (P < .001), 72.3 ± 16.9% at 3 months (P < .001), and 61.0 ± 34.4% at 5–6 months (P < .01). No complications or adverse events related to AM/UC injection were observed. These results suggest local perineural injection of AM/UC particulate may reduce pain and alleviate symptoms in patients suffering from painful peripheral neuropathy of the lower extremities.

ABOUT THE AUTHOR Ahmed Bilal Buksh, DPM, has more than 20 years of experience in podiatric medicine and peripheral nerve surgery. Dr. Buksh is board-certified in both podiatric surgery and podiatric medicine and his research focuses on addressing chronic pain using conservative and surgical methods. This research aims to support utilization of a novel conservative treatment option (i.e. human amniotic membrane and umbilical cord particulate) for patients with chronic peripheral neuropathy.

PUBLIC INTEREST STATEMENT
Peripheral neuropathy is primarily a disorder of the peripheral nerves that can result in pain, tingling, and numbness within the extremities. Current treatment options aim to relieve the symptoms without addressing the underlying cause and often fail to completely resolve symptoms with unfavorable side effects. In this work, symptomatic improvement was noted after perineural injection of AM/UC particulate without product-related adverse events. Perineural injection of AM/UC particulate resulted in significant relief of pain and associated symptoms at 1 month, 2 months, 3 months, and 5-6 months. These results suggest that AM/UC may be a viable treatment option for refractory cases.

Introduction
Peripheral neuropathy is primarily a disorder of the peripheral nerves caused by (1) compression due to space-occupying lesions (osteochondromas, tumors, ganglion/synovial cysts, tenosynovitis, varicosities), (2) compression secondary to shoes, osseous spurs or deformity, (3) traumatic injury, (4) compartment syndrome, (5) tissue hypertrophy, (6) stretching of the nerve or scar traction, or (7) systemic diseases (diabetes, peripheral vascular disease). In fact, painful peripheral neuropathy occurs in 25% of patients with diabetes treated in the office setting and 20-40% of cancer patients who receive neurotoxic chemotherapy (Spallone, Lacerenza, Rossi, Sicuteri, & Marchettini, 2012;Travis et al., 2014). Symptoms in the early onset include pain, paresthesia, and numbness within the extremities that subsequently have a negative impact on sleep, functionality, and quality of life (Iqbal et al., 2018). In advanced stages of the disease, there tends to be motor nerve dysfunction with distal weakness that may result in loss of balance or falling down (Feldman, Nave, Jensen, & Bennett, 2017;Pop-Busui et al., 2017).
Current treatment options are palliative, aiming to relieve the symptoms associated with neuropathy without addressing the underlying cause. Traditional therapies include over-the-counter pain relievers, opioids, anti-epileptic agents (pregabalin, topiramate, gabapentin), antidepressants, and muscle relaxants. Non-pharmacological treatments have also been used including low-intensity laser therapy, transcutaneous electrical nerve stimulation, and more commonly steroidal perineural injections. Nonetheless, these treatments often fail to completely resolve symptoms and can be associated with unfavorable side effects (Singh, Kishore, & Kaur, 2014). Consequently, chronic pain persists and leads patients in search of alternative therapies.

Materials and methods
After approval from the Institutional Review Board and waiver of informed consent, a retrospective medical chart review was conducted on patients (aged >18 years) that were diagnosed with peripheral neuropathy and received AM/UC particulate injection between June 2017 and July 2018 in accordance with the Declaration of Helsinki. All patients were treated by a single physician at a private practice. Patients were diagnosed with peripheral neuropathy if they were symptomatic, displayed decreased deep tendon reflexes, and had reduced sensation. Epicritic sensation was evaluated including sharp-dull, light touch, proprioception, pin-prick (at six points of the plantar surface), and protective threshold (10-g monofilament test). In addition, the 128 Hz tuning fork was applied for evaluation of vibration sensation, and provocative (pressure, Tinel's) tests were performed around the course of peroneal and tibial nerves. Diabetic induced neuropathy was confirmed with elevated A1c levels (≥6.5%). De-identified data were obtained from baseline (pre-injection) up to 1-year post-injection, including patient demographics (age, gender, ethnicity), type of neuropathy, neuropathy risk factors (glucose tolerance, pre-diabetes, diabetes mellitus, B 12 deficiency, hypothyroidism, immune disorder, and uremia), concurrent treatments, symptoms (e.g. pain, weakness, numbness, poor balance, reduced range of motion, and paresthesia in terms of numbness and tingling sensation), duration of symptoms, and complications.

Injection technique
All patients received ultrasound-guided injection at the common peroneal nerve at the knee, posterior tibial nerve at the ankle, and deep peroneal nerve on the dorsalateral foot. For injection of the common peroneal nerve (Figure 1), the patient was in the lateral decubitus position with the affected side up. The common peroneal nerve was found using a 7.5-to 15-Hz ultrasound probe (Sonoscape S9, Universal Medical Systems, Bedford Hills, NY) by scanning distally from the sciatic nerve in the proximal thigh and toward the proximal fibula. A diagnostic injection of 1cc of 1% lidocaine plain (Hikma Pharmaceuticals, London, United Kingdom) and 0.5cc of 8.4% sodium bicarbonate (Fresenius Kabi, Lake Zurich, IL) was performed for superficial anesthesia. Then, 25 mg of AM/UC particulate (CLARIX FLO®, Amniox Medical, Inc., Miami, FL) pre-mixed with 1.0 cc of 0.5% Marcaine plain (Fresenius Kabi, Lake Zurich, IL) in a 25-gauge, 1.5-inch needle was inserted via out-of-plane approach. The needle tip was positioned deep to the nerve to ensure adequate circumferential and proximal perineural distribution of the injectate outside the epinerium of the nerve. Needle repositioning was performed as needed to ensure complete coverage. Injection of the posterior tibial nerve at the ankle was performed with the patient in Fowler's position with the lateral foot down to expose the medial side of the hindfoot (Figure 2). The nerve was located using the ultrasound probe placed obliquely along the malleolar-calcaneal axis, with  the notch toward the medial malleolus and the other end directed toward the heel. After visualization of the tibial nerve, 3cc of local anesthetic (plain 1% lidocaine and 8.4% sodium bicarbonate) was injected. Thereafter, injection of 50 mg AM/UC particulate pre-mixed with 1.5 cc of 0.5% Marcaine plain (Fresenius Kabi, Lake Zurich, IL) was performed using a 22-gauge B-beveled block needle using in-plane technique. Lastly, injection of the lateral branch of the deep peroneal nerve was performed distal to the anterolateral part of the talus head and beneath the extensor hallucis brevis (Figure 3). Local anesthetic (1cc plain 1% lidocaine and 0.5cc 8.4% sodium bicarbonate) was first provided for superficial anesthesia. Then, injection of 1.0 cc of 25 mg AM/UC particulate/ Marcaine was performed to surround the nerve completely using in-plane ultrasound guidance with a 22-gauge, 50-mm-long Stimuplex needle. All patients remained in the office for 15 min after the injection to be monitored for adverse reactions. Afterwards, they were asked to avoid any strenuous activity for at least 24 h and ice the injected region as needed for post-injection pain up to 72 h. Patients were also asked to elevate the foot for 2 days and stop the use of antiinflammatory medications for a duration of 5 days post-injection.

Outcomes
At each follow-up visit, patients were asked to report their percent improvement in pain and associated symptoms compared to baseline before AM/UC treatment. Any unfavorable and unintended signs, symptoms, or disease spontaneously reported or discovered by the investigator during follow-up visits were reported as adverse events.

Statistical analysis
All statistical analyses were carried out using IBM SPSS version 20.0. Categorical variables were described by percentages and frequencies, while continuous variables were described by means and standard deviations. The Friedman test was used to assess whether improvement of symptoms was significantly different between time points. A P value <0.05 was considered statistically significant.

Results
A total of 17 patients met the eligibility criteria of this retrospective study and were included in the analysis. Patients (8 male, 9 female) had an average age of 50.5 ± 10.2 years and a mean BMI of 30.7 ± 6.4 kg/m 2 . Other baseline characteristics are summarized in Table 1. The diagnosis was diabetic (n = 8), idiopathic (n = 7), and chemotherapy-induced peripheral neuropathy (n = 2). The most commonly associated comorbidities included lower back pain (n = 11), diabetes (n = 8), and hypertension (n = 5). Autonomic nerves were damaged in two idiopathic neuropathy cases based on nerve conduction studies.
Patients received 2.7 ± 1.8 injections of AM/UC particulate per extremity. The average time between injections was 25.2 days for each extremity. No complications (including foot drop) or adverse events related to AM/UC were observed. However, one patient experienced vomiting 5-days post-injection and another patient fell in a parking lot the same day of injection.

Discussion
Patients with peripheral neuropathy often experience debilitating pain that significantly impacts their jobs, limits their social interactions, and impairs their day-to-day functionality. To alleviate the pain, these patients must weigh the benefits with the side effects of taking multiple pharmacological medication. Although patients might note a modest reduction in pain that allows a level of functionality that they would not have had otherwise, they might also experience a variety of cognitive side effects including drowsiness, difficulty focusing, and short-term memory loss. In the present study, patients presented with chronic symptoms that had been present for a median of 38.5 months, with 59% of patients experiencing motor weakness suggestive of chronic neuropathy progression. In addition, 94% of patients were refractory to prior perineural injections, and of those, many were not operative candidates due to various comorbidities. Nonetheless, patients showed symptomatic improvement after perineural injection of AM/UC particulate without product-related adverse events, suggesting that AM/UC may be a viable treatment option for refractory cases. Perineural injection of AM/UC particulate resulted in significant relief of pain and associated symptoms at 1 month, 2 months, 3 months, and 5-6 months in patients with diabetic (n = 8), idiopathic (n = 7), and chemotherapy-induced peripheral neuropathy (n = 2). In particular, the 8 patients with diabetic neuropathy in this study improved 50% at 1 month, 73% at 2 months, and 75% at 3 months with 2.75 treatments on average, which is promising as these patients are especially hampered in treatment options as steroids are known to cause hyperglycemia (Choudhry, Malik, & Charalambous, 2016).
Despite the favorable results, several limitations should be considered when interpreting the findings. First, the retrospective design only allowed analysis of data from prior medical records and not all data were consistently captured. Pain was not compared among individuals, rather measured before and after AM/UC for each individual. Patients were not followed up at exact specified timepoints and not routinely tracked for concomitant medication and compliance. Many of the patients were on nerve pain medications that may have influenced pain score. Future studies should track medication usage and laboratory values of vitamin B 12 and A1c that could confound symptoms. The AM/UC treatment was also not administered at consistent timepoints which introduces variability without a proper control group. With these limitations in mind, future studies could be designed with longer follow-up visits in order to study the duration of symptomatic relief.
If the aforementioned efficacy was verified in larger randomized studies, the underlying mechanism may be related to suppression of inflammation. AM/UC and its major biochemical component, i.e., HC-HA/PTX3, have been shown to significantly promote apoptosis of activated neutrophils (He et al., 2008;He, Zhang, Tighe, Son, & Tseng, 2013), promote phagocytosis by macrophages (He et al., 2013), and promote M2 macrophage polarization (He et al., 2009;He et al., 2013). These action mechanisms are advantageous in this particular application as nerve injury has been shown to activate inflammatory mediators and produce spontaneous neural activity (Moalem & Tracey, 2006). Hence, an analgesic effect is experienced after alleviating inflammatory response. Aside from this effect, AM/UC has also been shown to promote regeneration and normal neural regeneration which may also aid in pain alleviation (Morkin & Hamrah, 2017;Tseng, 2016). This action differentiates AM/UC from steroids, which are commonly used for perineural injections, but only provide an anti-inflammatory effect. Overall, perineural injection of AM/UC particulate may provide a differentiated alternative treatment option that aims to address the underlying pathologies.