Neuropathic pain in primary knee osteoarthritis patients: correlation with physical function, quality of life, disease severity, and serum beta nerve growth factor levels

Neuropathic mechanisms are thought to play a role in knee osteoarthritis (KOA) pain. Neuropathic pain questionnaires can promote diagnosis of a neuropathic component. Thus, we aimed to assess the frequency of neuropathic pain in primary KOA patients (using clinical questionnaires) and to investigate its correlation with socio-demographic factors, physical function, quality of life, disease severity, and serum beta nerve growth factor (β-NGF) levels. Seventy primary KOA patients were included. Neuropathic pain was detected in 52.9% of patients based on Douleur Neuropathique en 4 Questions (DN4) questionnaire and in 38.6% of patients based on Leeds assessment neuropathic pain symptoms and signs questionnaire (LANSS). Serum β-NGF levels were significantly higher in KOA patients than controls (P<0.0001), and in KOA patients with neuropathic pain compared with patients with non-neuropathic pain. DN4 score was positively correlated with Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain, stiffness, and physical function, and it was also negatively correlated with Osteoarthritis knee hip quality of life questionnaire (OAKHQOL) pain scores (rs=0.459, P<0.001; rs= 0.258, P= 0.031; rs= 0.307, P= 0.010; rs = −0.337, P= 0.004, respectively), while LANSS scale was positively correlated with symptom duration, WOMAC stiffness, Lequesne pain, and Lequesne index (rs= 0.260, P= 0.020; rs= 0.343, P= 0.004; rs= 0.344, P= 0.004; rs= 0.322, P= 0.007) and negatively correlated with OAKHQOL physical, OAKHQOL mental health, OAKHQOL social support, and total OAKHQOL scores (rs= −0.258, P= 0.031;rs= −0.254, P= 0.034; rs= −0.283, P= 0.018; rs= −0.261, P= 0.029 respectively). Neuropathic pain symptoms are frequent in primary KOA patients. KOA patients with neuropathic pain have worse quality of life, extreme disability, and higher serum β-NGF levels. Nerve growth factor inhibitors could have a potential role for not only relieving pain in KOA patients but also improving functional disability and quality of life in these patients.


Background
Osteoarthritis (OA) is a multifactorial disease, involving multiple causative factors as aging, trauma, biomechanical loading, inflammation, and metabolic disturbances [1,2]. It is classified into two groups according to its etiology: primary (idiopathic or nontraumatic) and secondary (usually due to trauma or mechanical misalignment or other articular disease). The disease is more common in middle-aged and older people over the age of 50. The prevalence of knee osteoarthritis (KOA) among female patients is higher than that of male patients, and the prevalence rate of individuals aged over 65 years is 68% [3].
Pain and local tenderness or pressure hyperalgesia around the affected joint are frequently manifested in OA [4]. OA pain mechanisms are complex; both peripheral and central processes are involved in creating the OA pain [5].
People with KOA may present with different pain phenotypes, nociceptive, inflammatory, and neuropathic pains [5,6]. Neuropathic component of OA pain, possibly emerging from peripheral and central pain sensitization mechanisms [7]. Recognizing specific aspects of joint pathology that contribute to different OA pain phenotypes might help identify pain phenotype specific peripheral treatment targets [8].
Several screening tools were used to differentiate neuropathic pain from non-neuropathic pain [9]. All screening tools have self-assessment questions. However, sensory examination is present in Leeds assessment of neuropathic pain symptoms and signs (LANSS) and Douleur Neuropathique 4 questions (DN4) questionnaires, which give them an objective significance and crucial findings for the diagnosis of neuropathic pain, among all the others [10].
Nerve growth factor (NGF) is a key mediator of acute and chronic pain. Different biological actions of NGF contribute to its pro-analgesic effects, including NGFinduced sensitization of peripheral nociceptive terminals and NGF-induced sprouting of sensory nerves [11]. Nerve growth factor binds tropomyosin receptor kinase A (TrkA) that is expressed in many sensory and sympathetic fibers and regulates survival of these neurons [12]. Elevated NGF levels are seen in individuals with chronic pain conditions [13], and intradermal or intramuscular injection of NGF causes allodynia and hyperalgesia in healthy subjects [14,15].
The purpose of this study was to determine the frequency of neuropathic pain in patients with primary KOA (using the DN4 and LANSS questionnaires) and to investigate its correlation with socio-demographic factors, physical function, quality of life, and disease severity. Despite the fact that serum-NGF levels are increased in KOA patients, their probable links to neuropathic pain have never been investigated. Therefore, we aimed to assess serum β-NGF levels in KOA patients with neuropathic pain versus patients with non-neuropathic pain.

Study design and population
This study used an analytical cross-sectional design. Seventy patients (43 females and 27 males) with primary KOA who met ACR clinical and radiographic diagnostic criteria for primary KOA [16] were consecutively included. All patients were attending the Rheumatology and Rehabilitation outpatient clinic, Minia University Hospital, Minia governorate, Egypt-in the period from December 2018 to March 2019. All patients had knee pain for more than 3 months. The control population consisted of 21 (13 females/8 males) age-, sex-, and body mass index (BMI)-matched healthy volunteers (who are healthcare workers at the Main Hospital/for assessment of serum β-NGF levels) without any sign or disease suggestive of OA, gout, rheumatoid arthritis, trauma, diabetes, and other orthopedic disorders like tendinitis, bursitis, and knee ligament injuries. Written informed consent was taken from all participants in the study. The study was approved by the ethics committee of the Faculty of Medicine. We had to enlist the help of research assistants who met with each illiterate patient and read out the questions in a way that they could understand, without necessarily affecting their responses.
Excluded from the study are patients with any previous history of knee surgery, history of steroid injections over the past 3 months, trauma, infection, known inflammatory arthritis such as rheumatoid arthritis, gout and other pain/neurological conditions such as radiculopathies, diabetes mellitus, stroke, and traumatic brain injury and patients who were already receiving medical treatment for neuropathic pain.

Clinical assessment Anthropometrics
Height was measured to the nearest 0.1cm, weight was measured in the upright position to the nearest 0.1 kg, and BMI is calculated as weight in kilograms divided by the square of height in meters.

Visual analogue scale
Visual analogue scale (VAS) is a horizontal line, 100 mm in length, anchored byword descriptors at each end. The patient marks on the line the point that they represented their perception of their current state. A higher score indicates greater pain intensity [17].

Western Ontario and McMaster University (WOMAC) Osteoarthritis Index
The WOMAC is a valid and reliable instrument used for the assessment of OA of the lower extremities [18]. It consists of 24 items divided into 3 subscales. The pain scale includes five items asking about pain at activity or rest. The stiffness scale includes two questions. The function dimension explores the degree of difficulty in daily activities. The scores are summed for items in each subscale, with possible ranges as follows: pain=0-20, stiffness=0-8, physical function=0-68, and total WOMAC score is created by summing the items for all three subscales (0-96).

Lequesne index
Lequesne index was used to evaluate severity for knee disease. It is eleven-question survey, five questions pertaining to pain or discomfort, two questions dealing with maximum distance walked, and four questions about activities of daily living. The total questionnaire is scored on a 0 to 24 scale. Lower scores indicate there is less functional impairment [19].
Osteoarthritis knee hip quality of life questionnaire (OAKHQOL) The patient's health-related quality of life was assessed using the OAKHQOL questionnaire, including 43 items in five main domains: physical activities (16 items), mental health (13 items), pain (4 items), social support (4 items), social functioning (3 items), and three independent items; each item is scored on a scale from 0 to 10, and the normalized scores were obtained by computing the sum of item scores for each domain and calculated to a scale from 0 (worst) to 100 (best) [20]. We had to enlist the help of research assistants who met with each illiterate patient and read out the questions in a way that they could understand, without necessarily affecting their responses.

Evaluation of neuropathic pain
Detailed history taking, neurological examination, and two standardized screening tools were performed on all KOA patients. Screening tools were used for the purpose of distinguishing neuropathic pain from nonneuropathic pain.

Douleur Neuropathique 4 questions (DN4 questionnaire)
It was developed and validated in French and translated into 15 languages. It consists of 10 items that are either answered as YES or NO. Seven of these items assess pain quality, while the other 3 items detect the presence or absence of sensory allodynia and touch needle hypoesthesia based on clinical examination. Each item answered as "yes" yields 1 point, and a total score at or above 4/10 is evaluated as positive [21]. Arabic version of DN4 questionnaire was used [22].

Leeds assessment of neuropathic pain symptoms and signs (LANSS scale)
The LANSS scale is a valid tool used for discriminating between neuropathic and nociceptive pain. It contains 5 symptom items and 2 clinical examination items, score ≥ 12 suggest likely neuropathic pain [23]. Arabic version of LANSS scale was used in the present study [24].

ELISA for serum β-NGF
Five millimeters of venous blood was withdrawn, collected in serum separator tubes, and allowed to clot for 10-20 min at room temperature. Centrifuge at 2000-3000 RPM for 20 min and stored at −80°C for later use. Serum β-NGF levels were measured for patients and healthy controls using an enzyme-linked immunosorbent assay (ELISA) kit (SHANGHAI CRYSTAL DAY BIO-TECH CO., LTD, China). Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.

Statistical analysis
The statistical analysis was performed using SPSS 22.0. Descriptive statistics were done by number and percent as well as mean and SD. Statistical differences between groups were tested using chi-square (χ 2 ) test for qualitative variables and independent sample t-test for quantitative normally distributed variables. Correlations were calculated using Spearman's correlation coefficient. Linear regression analysis was used to predict the risk factors for neuropathic pain in primary KOA patients. The level of statistical significance was set at a P ≤ 0.05.

Results
Sociodemographic data of the studied population are shown in Table 1 Patients who had scores ≥4 from the DN4 questionnaire and scores ≥12 from the LANSS scale were accepted to have neuropathic pain. According to this, neuropathic pain was detected in 37 patients (52.9%) based on the DN4 questionnaire and in 27 patients (38.6%) based on the LANSS scale. DN4 scale ranged between 2 and 8 with a mean of 4.39± 1.51 while LANSS score ranged between 9 and 19 with a mean of 12.2±3.03.
The frequency of responses for individual items of DN4 questionnaire and LANSS questionnaire among OA patients is shown in Tables 2 and 3.
Comparison of WOMAC scores, Lequesne scores, Kellgren-Lawrence scale, and normalized OAHKOQL scores between patients with neuropathic pain and those with non-neuropathic pain is shown in Tables 4 and 5.
The mean serum βNGF levels were significantly higher in KOA patients than control: 590.7 ± 244.83 pg/ml versus 27.52 ± 6.87 pg/ml respectively, P <0.0001. Moreover, the mean serum βNGF levels were significantly higher in patients with neuropathic pain than patients with non-neuropathic pain as shown in Table 5.
The correlations of neuropathic pain scores with all of the studied parameters are shown in Table 6.
Linear regression analysis was employed to analyze the risk factors for neuropathic pain in primary knee OA. According to DN4 score, higher WOMAC pain, higher WOMAC physical function, higher total WOMAC, and higher βNGF were significant risk factors (P<0.001, P< 0.001, P= 0.002, P= 0.039 respectively). While according to LANSS score, longer symptom duration, higher WOMAC stiffness, higher Lequesne pain, lower social support OAKHQOL, lower total OAKHQOL, and higher serum βNGF level were significant risk factors for neuropathic pain (P= 0.026, P <0.001, P= 0.048, P= 0.019, P=0.036, P < 0.001 respectively).

Discussion
In the present study, neuropathic pain was detected in 52.9 % of patients based on the DN4 questionnaire and in 38.6 % of patients based on the LANSS scale. According to DN4 questionnaire, the most frequently described neuropathic pain characteristic in KOA patients with neuropathic pain was a sensation of electric shock (48.7%) with a significant difference compared to KOA patients with non-neuropathic pain, and on physical examination up to 30% had touch hypoesthesia, while    according to LANSS scale the most frequently knee pain quality symptom was pins and needles tingling or pricking (35.7%) followed by burning pain (34.3%) with a significant difference compared to KOA patients with nonneuropathic pain; however self-exam items revealed that 24.3% of patients had numbness or tenderness which felt when pressing on the painful area with a significant difference compared to patients with non-neuropathic pain and 14.3% had pins and needles, tingling or burning on rubbing on the painful area.
Aşkın et al. [31] refereed the wide variation in the reported prevalence of neuropathic pain in OA to differences in methodology between studies and pain assessment tools.
Our results revealed that KOA patients with neuropathic pain as detected by DN4 questionnaire have significantly higher WOMAC pain, WOMAC physical function scores, and significantly lower normalized pain OAKHQOL score than patients with non-neuropathic pain (P <0.0001, P= 0.015, P =0.018 respectively). Moreover, significantly higher Lequesne pain score and Lequesne index were found in KOA patients with neuropathic pain as detected by LANSS scale compared with patients without neuropathic pain (P=0.036, P=0.022 respectively).
In consistent with our results, Gölge et al. [27] found a highly significant difference between neuropathic and non-neuropathic groups as regards WOMAC pain score (p <0.001), Aşkın et al. [31] also found a significant difference between both groups as regards WOMAC physical function (p = 0.04), Narayan et al. [32] found a significant difference between both groups as regards WOMAC total score (p =0.024) and WOMAC physical function score (p = 0.008), Mahmoud et al. [38] study showed that the total score and normalized pain domain score were worst in the neuropathic group than the non-neuropathic group, and Radwan and Borai [33] found a highly significant difference between neuropathic and non-neuropathic pain groups as regards WOMAC physical function score, WOMAC pain score, and WOMAC total score (p < 0.001 for all of them).
We assessed serum β-NGF levels in the present study and we have found a significantly higher serum β-NGF levels in KOA patients than controls (P<0.001), and significantly higher serum β-NGF levels in KOA patients with neuropathic pain "as detected by DN4 questionnaire and LANSS scale" compared with patients without neuropathic pain (P= 0.01, P= 0.004 respectively). In agreement with our results, a study of Montagnoli et al. [39] reported significantly higher serum and synovial β-NGF levels in KOA patients than controls. To our knowledge, no previous studies in the literature assessed serum β-NGF levels in KOA patients with neuropathic pain versus patients without neuropathic pain.
A number of evidences indicate that β-NGF plays a significant role in osteoarthritis, not only in pain and hyperalgesia by nociceptor sensitization, but also as a key element of the inflammatory process [40,41]. Neuropathic pain is unresponsive to common analgesics, such as NSAIDs. Systemic central acting drugs as duloxetine, an antidepressant, have proven effective in controlling this type of pain in OA [42]. Agents blocking NGF might have therapeutic utility for pain [43].
Female sex, age, and BMI are well-known risk factors for OA, as shown in previous studies [44][45][46]. Also, a low level of education was found to be a significant factor associated with OA [47]. In the present study, we have found no correlation between neuropathic pain scores and risk factors. Study of Polat et al. [37] was consistent with our findings. In contrary to our results, Hochman et al. [35] found that the patients with neuropathic pain were younger and were more likely to be females, but there was no significant difference in level of education between patients with neuropathic pain and those without neuropathic pain.
Our results revealed no correlation between neuropathic pain scores and the Kellgren-Lawrence grades and this was consistent with the previous results of Narayan et al. [32], Polat et al. [37], and Radwan and Borai [33]. In contrary to our results, Ohtori et al. [36] reported that neuropathic pain tended to be seen in patients with KL grades of late stages OA; however, the majority of our patients had mild to moderate OA.
In the present study, according to linear regression analysis, longer symptom duration, higher pain scores, lower OAKHQOL score, and higher serum βNGF levels were considered as significant risk factors for the development of neuropathic pain in KOA patients.
Our study has some limitations. Firstly, this study was cross-sectional with a relatively small number of patients. Secondly, we have not assessed for central sensitization by measuring pain pressure thresholds. Lastly, we did not assess β-NGF levels in synovial fluid and/or synovial tissue.

Conclusions
Neuropathic pain is frequent in KOA patients. Our study is the first one that assessed serum β-NGF levels in Egyptian primary KOA patients, and studied its correlation with the presence of neuropathic pain in OA. We recommend further studies which include larger number of KOA patients, in order to classify patients according to β-NGF levels, and further investigations to assess the role of β-NGF antagonists in relieving pain and improving functional status and quality of life in KOA patients.