Caraway Oil as a Multimodal Therapy for Neuropathic Pain: Investigating the Mechanisms of Action in Rats with Chronic Constriction Injury

Abstract

Neuropathic pain, a prevalent concern associated with various pathological conditions, poses a significant public health risk due to its poorly understood pathophysiology and treatment complexities. Multimodal therapy is often the most efficacious approach to managing neuropathic pain, yet it is also highly labour intensive. The exact underlying causes of neuropathic pain are unclear; evidence suggests that cytokines, neuropeptides, and neurotrophic factors may play a role in its pathogenesis. The current study aimed to investigate the anti-neuropathic pain activity of caraway oil and the molecular mechanisms underlying its actions in rats with CCI, a model of neuropathic pain. Behavioural evaluations of cold allodynia, heat hyperalgesia, mechanical allodynia, and mechanical hyperalgesia were conducted using the acetone spray test, hot plate test, Von Frey hair test, and pinprick test, respectively. Additionally, the level of TNF-α in the sciatic nerve was examined as an indicator of inflammation, and NGF and substance P levels were determined in the DRG to identify mechanistic processes. Rats were administered caraway oil orally at doses of 25 and 50 mg/kg for 21 days. Results indicated that caraway oil administration significantly reduced behaviour associated with injury-related pain and elevated TNF levels. After an anti-NGF injection on the 21st day, significant attenuated behavioural effects were observed. Furthermore, caraway oil administration was able to inhibit the upregulation of NGF in DRG caused by CCI and minimize the increase in substance P in DRG. These findings suggest that caraway oil has promising therapeutic potential for managing neuropathic pain by targeting peripheral and secondary sensitization mechanisms.

1. Introduction

Neuropathic pain is a complicated disorder that can be a long-lasting complex chronic condition characterized mainly by various sensory, cognitive, and emotional symptoms [1]. An abnormal alteration of the somatosensory system, resulting from damage to the nervous system, is the real cause of neural pain [2]. The senses of touch, pressure, pain, temperature, position, motion, and vibration are all perceived through the somatosensory system. In total, 7–10% of the general population is affected by the neuropathological pain from the somatosensory system, which includes central neurons and peripheral fibres (Aβ, Aδ, and C fibres). Several factors that can lead to neuropathic pain and its pervasiveness are expected to increase over time due to factors such as the ageing of the world population, the increasing prevalence of diabetes, and higher cancer survival rates after chemotherapy [3].

Neuropathic pain is caused by nerve damage in the central and peripheral nervous systems [4]. Worldwide, peripheral neuropathic pain is becoming increasingly debilitating, with an increasing number of people suffering from it [5]. After peripheral nerve damage, a series of actions occur in the primary afferents that result in peripheral sensitization, leading to impulsive nociceptor activation, increased reactivity to suprathreshold stimuli, and lowered pain threshold [6]. The inflammatory reactions in the injured nerve trunk may cause ectopic activation of nociceptors, which would result in spontaneous pain. During overactive nociceptors, the brain and spinal cord undergo pathological changes. These changes contribute to the perception of mechanoreceptive fibres as painful sensations. A constant supply of growth factors on normal skin is essential for maintaining the neuronal phenotype [5].

It is well-established and well-supported that nerve growth factor (NGF) is associated with different pains, including visceral, inflammatory, and neuropathic [7]. In both the peripheral and central nervous systems, NGF is a vital neurotrophin that controls neuron survival and development [8]. NGF is retrogradely transported from the innervation sites of sensory neuron cell bodies, which modulate neuropeptide transmitter concentrations [9]. In the animal model, Waller’s degeneration is a prerequisite for neuropathic pain after peripheral nerve injury. Activated Schwann cells produce significant amounts of NGF over many weeks during Waller’s degeneration; thus, in the context of nerve diseases, NGF may play a role in neuropathic pain [10]. McKelvey et al., in 2013, concluded that various pains and conditions had been associated with elevated levels of growth factor NGF and its involvement in enhancing pain sensation. The analgesic effects of NGF antagonism have the potential to be improved compared with currently available treatments [11]. In addition, pro-inflammatory cytokines have been found to increase pain sensations by altering neurotransmitter release, particularly substance P [12]. In the peripheral and central nervous systems, the substance P is known as a transmitter of nociception and inflammation by altering cellular signalling pathways [13]. Furthermore, the literature disclosed the contribution of inflammatory cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase, and nuclear factor kappa B (NF-κB) in the pathogenesis of neuropathic pain [12].

There are several hypotheses about peripheral neuropathic pain and its treatment, but we have to give attention to the factors that cause the condition or symptoms per se, and the processes that cause the transformation of such conditions [14]. Most research has focused on conventional non-steroidal anti-inflammatory drugs and opioid-based treatments [15]. Some conventional analgesics, such as carbamazepine, pregabalin, gabapentin, etc., have been accepted for treating neuropathic pain for the last 50 years. However, the prospects for pharmacological management persist in being inadequate and are incorporated with restricted efficacy and pivotal consequence, as most of them accommodate nonspecific signs of activity-related pain alleviation related to the administration of neuropathic therapy [16]. Despite current research to understand the peripheral and central sensitization pathways associated with nervous system injury, treating neuropathic pain remains a therapeutic challenge. There is no widespread consensus on which method is most effective in relieving neuropathic pain [17]. In recent years, the issue of neuropathic pain has come to the forefront more often, and it is now regarded as one of the most significant difficulties facing contemporary medicine [18]. As a result, there is still a significant need to explore innovative therapeutic methods and carry out mechanistic studies to treat neuropathic pain. In our current research, we strive to understand the pathophysiology of neuropathic pain better. We are particularly interested in identifying the processes contributing to pain and the therapies successfully targeting these mechanisms.

In recent years, there has been a growing belief that medicinal plants are a safer treatment option over synthetic drugs. This change has led to the increasing use of phytopharmaceuticals [19]. Caraway (Carum carvi L.) belongs to the umbellifers family and is native to Europe, Asia, and North Africa. Seeds are used as spices in foods due to their pleasant taste [20]. Caraway essential oil has antioxidant [21], digestive and IBS-preventative [22], antidiabetic [23], antihyperlipidemic, weight-lowering [24], anticancer [25,26], antimicrobial [27,28,29] and anti-inflammatory [30,31,32] potential. Caraway oil is available in various marketed formulations with anti-inflammatory, antibacterial, and other effects, alone or in combination with others [31,33,34]. The knowledge gained from this literature can then be used to verify the presumed medicinal effects of caraway oil. However, the anti-neuropathic effect of caraway oil on chronic constriction injury (CCI) has not been investigated. Therefore, the present study was designed to explore the effect of caraway oil on CCI-induced neuropathic pain in rats and to establish the relationship between the inhibition of NGF and the substance P in the observed effect. The summary of the work is presented as graphical Scheme 1.

2. Results

2.1. Effect of Caraway Oil on Hyperalgesia and Allodynia in CCI-Induced Neuropathic Pain

CCI resulted in the development of cold allodynia (Figure 1), mechanical allodynia (Figure 2), mechanical hyperalgesia (Figure 3), and heat hyperalgesia (Figure 4), especially compared to the sham group, as evaluated by the acetone sprinkler test, Von Frey hair test, pinprick test, and hot plate test, respectively. On days 7 and 21, CCI-induced hyperalgesia and allodynia were significantly lower when caraway oil (25 and 50 mg/kg) was administered for 21 days. However, compared to days 7 and 21, there was a significant increase in discomfort sensations on day 21. Caraway did not affect the behaviour of normal rats when it was given at dosages of 25 and 50 mg/kg. Both hyperalgesia and the allodynia in group VI that CCI generated were not affected by vehicle administration.

2.2. Effect of Caraway Oil on TNF-α and Total Protein Content in CCI-Induced Neuropathic Pain

Unlike sham-operated animals, CCI increased TNF-α levels in the sciatic nerve on the 21st day. But after the administration of 25 mg/kg and 50 mg/kg of caraway oil, there was found a significant reduction in the TNF-α levels on the 21st day (Figure 5). There was no significant difference observed in the level of total protein content.

2.3. Effect of Caraway Oil on NGF and Substance P in CCI-Induced Neuropathic Pain

In the CCI subgroup, anti-NGF therapy (3 µg/50 µL) significantly decreased from the first to the fifth hour after injection. Anti-NGF (3 µg/50 µL) was most effective in diminishing thermal hyperalgesia 2 h after administration. Five hours after anti-NGF administration, the effect of anti-NGF was found to be evident in all behavioural evaluations. Anti-NGF medication reduces NGF upregulation in the DRG, and this reduction was statistically significant compared to sham groups. Compared to CCI + vehicle, substance P- a neuropeptide decreased in the DRG following anti-NGF therapy (3 µg/50 µL). The expression level of substance P in the anti-NGF group was relatively lower than the NGF expression level (Figure 6).

3. Discussion

The CCI model is the most widely used animal model for investigating neuropathic pain, described by allodynia and hyperalgesia caused by nerve damage (Zhao et al. 2017, Chen, Hu et al. 2018). This model mimics human carpal tunnel syndrome, in which the median nerve is compressed as it passes through the carpal tunnel by a swollen ligament or inflammation of the tendons within the tunnel. Additionally, neuropathological pain is caused by the encapsulation of the sciatic nerve by four loose ligatures. Furthermore, it has been proposed that this model mimics the pathophysiology of complex regional pain syndromes in people [35]. The results showed that cold allodynia, mechanical allodynia, mechanical hyperalgesia, and heat hyperalgesia increased significantly on day 7 after CCI. In the CCI model, significant behavioural changes were observed on day 21 in all treatment groups.

Caraway oil was administered orally at 25 mg/kg or 50 mg/kg in rats for 21 days. As a result, CCI-induced behavioural abnormalities, such as cold allodynia, mechanical allodynia, and mechanical heat hyperalgesia, were considerably reduced. Several pharmacological investigations have established the therapeutic use of caraway oil in various inflammatory circumstances, along with a rat model of chronic ischemia in cerebral palsy, acute lung damage after cecal ligation, and puncture-induced polymicrobial sepsis in rats [31,36]. However, it has been hypothesized that caraway oil can help with CCI-induced neuropathy in rats. In this study, the therapeutic benefit of caraway oil was demonstrated in the CCI model when it was given to rats for 21 days.

The study findings showed that increased TNF-α levels in the sciatic nerve were related to the neuropathic pain produced by CCI. TNF-α is the prototypical example of a pro-inflammatory mediator because of its location at the beginning of the cytokine cascade. It is widely recognized that TNF-α plays a role in neuropathic pain by making nerves in the peripheral and central nervous systems more sensitive. This fact has been demonstrated by an extensive research [35]. The findings of this study, which demonstrate higher levels of TNF-α in the sciatic nerve on day 21 in rats that had been treated with CCI, align with the findings of other investigations [37]. The decrease in TNF-α levels could have been caused by suppression of substance P, nerve growth factor, or cytokines. Several studies have shown that cytokines involved in inflammation, such as IL-1β, TNF-α, and IL-6, are promoters of NGF synthesis in different cells [38]. Caraway oil has a significant impact on suppressing neuropathic pain in rats. This is likely because it mediates a decrease in cytokine production, especially of TNF-α. Carvone (a significant constituent of caraway oil) decrease the biosynthesis of leukotrienes, prostaglandins, and inflammatory cytokines (IL-1, TNF-α) [39].

In this study, a single dose of anti-NGF antibody blocked endogenous NGF, which helped relieve pain in a model of chronic neuropathy. The model was used to study this phenomenon. In rats with CCI, anti-NGF treatment dramatically reduced mechanical and thermal hyperalgesia and cold allodynia. Very few findings have been published on the effectiveness of CCI in combination with anti-NGF therapy for treating chronic pain in rats. Consequently, processes involved in pain relief have not been identified [7]. The present study used behavioural analysis, NGF, and substance P in DRG to show how effective anti-NGF is in reducing chronic neuropathic pain.

The substance P that is released in the dorsal horn of the spinal cord is known to participate in the process of modulating pain and transmitting pain signals. There is a correlation between increased secretion and the presence of physical discomfort and acute stress [40]. The release of substance P occurs in response to inflammation in the periphery and noxious stimuli. Substance P plays a crucial role in nociception at the spinal level. Substance P can be secreted in the dorsal horn in response to ordinarily ineffective benign stimuli in rats with behavioural hyperalgesia caused by experimental polyarthritis. After an inflammatory response, the superficial dorsal horn of the spinal cord has elevated levels of NK1 mRNA expression [41]. In the current investigation, we observed the same categories of effects caused by substance P.

Furthermore, anti-NGF treatment inhibited the upregulation of NGF in DRG, as well as substance P in DRG in response to neuropathic pain. It is hypothesized that the analgesic impact of anti-NGF in chronic neuropathic pain is due to its direct influence on peripheral sensitization by lowering NGF and substance P and its indirect effect on central sensitivity by reducing the activation of the anterior cingulate cortex. This is because anti-NGF has both a direct and an indirect effect on peripheral sensitization. The anterior cingulate cortex may be responsible for the reported brain effect by relaying projections to the spinal cord without affecting synaptic transmission to the grey periaqueductal cortex. This is a possible mechanism that could have caused the effect. In conclusion, caraway oil alleviates the symptoms of neuropathic pain in a CCI model. This could be due to the fact that caraway oil can inhibit the production of pro-inflammatory mediators that are triggered during nerve injury.

4. Materials and Methods

4.1. Chemicals

Caraway oil was obtained from Sigma-Aldrich Chemicals, Bangalore. India. All reagents were obtained from S.D. Fine, Mumbai, India, with an analytical grade. Polyclonal rat β-NGF antibodies (anti-NGF), tumour necrosis factor (TNF)-α assay kits, and anti-substance P antibodies produced in rabbits were purchased from Merck Chemicals.

4.2. Experimental Animals

Sprague Dawley female rats weighing 190–250 g were used in the current study, as shown in Scheme 2. The research was approved by the Standing Committee on Bioethics of Prince Sattam Bin Abdulaziz University (SCBR-024-2022), Al-Kharj, Ministry of Education, Kingdom of Saudi Arabia. Light and dark cycles and relative humidity and room temperature were carefully monitored and controlled for the rats at 25 ± 1 °C. The availability of food and water was not restricted for the rats.

4.3. Induction of Neuropathy by CCI

CCI induced peripheral neuropathy in the same manner as described by De Vry et al. (2004) but in company with slight alterations. The sciatic nerve is exposed in the middle of the upper arm and is tied with silk sutures [42]. Additionally, silk sutures cause inflammatory reactions in the sciatic nerve. The dose of pentobarbital sodium administered intraperitoneally to rats was fixed at 50 mg/kg. Prior sutures were applied, and the lower back and thighs of the rats were shaven and thoroughly cleaned with 0.5% chlorhexidine. The epidermis was cut open to access the sciatic nerve on the left side of the leg, and a surgical incision was made right through the biceps femoris muscle. After the nerve was exposed, it was ligated to the surrounding tissue at four different sites, each site about 2–3 millimetre apart. The ligatures were loosely knotted until a transient twitch was observed in the ipsilateral hindlimb. Two layers of sutures were used to seal the wound in the muscles and skin, and topically applied antibiotics were used. Following the appropriate sterility conditions, all surgical procedures were performed.

Group I (Normal Control)—Rats were treated with vehicle (0.5% CMC) for 21 days. Group II (Sham control)—On day 1, the sciatic nerve was exposed during surgery performed on rats. No nerves were ligated during this procedure. Group III (CCI)—Rats were not treated for 21 days. Group VI (Caraway oil 25 mg/kg) and Group V (Caraway oil 50 mg/kg)—Caraway was administered orally at a dose of 25 mg/kg and 50 mg/kg with 0.5% CMC to rats undergoing CCI, beginning on day 1 (30 min before anaesthesia for surgery) to day 21. Behavioural tests were performed on day 1 before surgery, day 7, and day 21. Group VI (Vehicle-treated rats with CCI)—CCI in rats treated with vehicle. Rats treated with CCI received vehicle (0.5% CMC; 1 mL/kg) administered orally from day 1 to 21 (30 min before general anaesthesia for surgery). Group VII (anti-NGF with CCI)—Anti-NGF (received 3 μg/50 μL) was administered via an intraplantar route into animals on day 21. Behavioural tests were performed on day 0, day 7, and day 21 (after 1 h of anti-NGF administration). On day 21, the animals were sacrificed, the dorsal root ganglia (DRG) were removed, and biochemical parameters were estimated.

4.4. Behavioural Studies

4.4.1. Determination of Paw Cold Allodynia (Acetone Sprinkling Test)

To determine whether or not the rat had cold allodynia, 100 µL of acetone was applied externally on the surface of the rat’s paw (which was supported by a wire mesh), but it did not come into contact with the skin. Following the method developed by Flatters and Bennett (2004), the influence of acetone was examined for twenty seconds and scored on a four-point scale. Acetone was applied thrice to the hind paw, with a gap of 5 min between the acetone applications. Over a full minute, the 20 s intervals were averaged, and a single point was calculated from them. The lowest possible score was zero, and the highest was nine [43]. A score of 0 indicated no response, 1 was quick withdrawal or flicking, 2 was prolonged withdrawal or repeated flicking of the paw, and 3 was repeated flicking of the paw with licking of the paw. Cumulative scores were then obtained by adding the three scores for each rat.

4.4.2. Determination of Mechanical Hyperalgesia (Pinprick Test)

To determine the presence of mechanical hyperalgesia, Erichsen and Blackburn-Munro (2002) developed a pinprick test [44]. At a 90° angle to the syringe, the tip of a curved measuring needle was stuck to the injured hindfoot. The pressure was so strong that the animal reflexively pulled away from the touch. The time it took for the paw to pull back was measured in seconds, and the normal rapid response to reflexive withdrawal was reported to be 0.5 s.

4.4.3. Determination of Paw Heat Hyperalgesia (Hot Plate Test)

The Eddy heat plate was evaluated at a temperature of 52.5 ± 1.0 °C to assess the nociceptive heat threshold as an index of thermal hyperalgesia. On the hot plate, the rat was placed, and the time it took to lick the hind paw was recorded in seconds. The cut-off time of 25 s was maintained [45,46].

4.4.4. Determination of Mechanical Allodynia by Von Frey Hair Test

Following the method described by Chaplan et al. (1994), we tested mechanical allodynia with noncorrosive mechanical stimuli [47]. The central plantar surface of the left hind paw was treated with calibrated nylon threads, which were then placed in several shapes. The filaments were layered ten times, beginning with the most flexible filament and working up to the most rigid. A good response was considered given when the left hind leg was rapidly retracted. The criterion for the threshold value, in grams, was equal to the filament evoking a withdrawal threshold of the left hind paw five times out of 10 trials, i.e., a 50% response. If the rodent withdraws, licks, or shakes their paw, they are considered to have had a positive response. Rats were scored as 0 (no response), 1 (withdraws, licks), or 2 (vigorous licks or shakes the paw), and the scores were summed to yield a total allodynia score.

4.5. Biochemical Estimations

All animals were euthanised with cervical dislocation, and samples of their sciatic were taken. The biochemical investigation of this was started right away once it was extracted. After homogenising the sciatic nerve in phosphate-buffered saline with a pH of 7.4, the resulting homogenates were centrifuged at 4 °C for ten minutes at an RPM of 1500 per g. Furthermore, supernatants were collected to analyse the protein concentration and TNF-α [35]. Utilizing bovine serum albumin as a standard, the protein content in the sciatic nerve homogenates was estimated with Micro Lowry, Peterson′s method with modification using total Protein Kit (CAS-TP0300, Merck KGaA, Darmstadt, Germany) [48,49].

4.5.1. Estimation of TNF-α

TNF-α was determined with a commercially feasible ELISA kit (CAS-RAB0480, Merck KGaA, Darmstadt, Germany). The TNF-α was evaluated regarding picograms per milligram of protein.

4.5.2. Estimation of NGF and Substance P

To determine the level of NGF expressions, the ELISA kit (CAS-RAB0381, Merck KGaA, Darmstadt, Germany) was used. The substance P expression was also estimated using an ELISA kit (CAS-S1542, Merck KGaA, Darmstadt, Germany) carefully following the manual’s instructions.

4.6. Statistical Analysis

The findings were presented as mean values with standard error means (SEM). GraphPad Prism version 5.0 was used to look at the results of the behavioural tests. First, a two-way analysis of variance (ANOVA) was performed, and then a Tukey multiple range test was performed. A p-value ≤ 0.05 was necessary for a finding to be statistically significant.

5. Conclusions

Nerve damage can cause persistent discomfort, increased sensitivity to even the lightest touch (allodynia), and the loss of sensation where injured and uninjured nerves converge. Chronic pain and allodynia can also be caused by nerve damage. Despite the limited therapeutic options available, neurogenic pain is a severe condition that negatively influences the quality of life of individuals who suffer from it. Due to its prevalence, severity, and debilitating effects, chronic neuropathic pain is a significant public health burden. Despite various pharmaceutical options, there is currently no conventional treatment that can eliminate neuropathic pain. Due to the complex mechanisms involved in developing neuropathic pain, the treatment of neuropathic pain continues to be a significant obstacle in clinical practice. Recent years have seen an increase in the amount of focus placed on herbal remedies within the pharmaceutical industry.

Our results indicate that caraway oil, when administered orally, alleviates the symptoms of neuropathic pain caused by CCI in rats. It is possible to make relevant conclusions about the use of caraway oil as an alternative medicine for the treatment of neuropathic pain based on the findings of this study, which are supported by evidence. According to the findings of our study, the application of caraway oil to a rat model of neuropathic pain prevented the activation of both NGF and substance P. Furthermore, it reduces the levels of an inflammatory cytokine (TNF-α).

Caraway has been shown to have anti-neuropathic effects. Still, more study in various animal models is necessary to determine its precise mechanism of action in anti-NGF treatment. The validation of the data gives us optimism for future success in treating neuropathic pain with fewer adverse effects. The oral administration of caraway oil has been suggested as a potential therapeutic approach for treating neuropathic pain.

Author Contributions

Conceptualization, F.K.A. and S.D.; methodology, S.D.; software, H.S.Y.; validation, F.K.A. and A.A.; formal analysis, A.A.; investigation, S.D.; resources, F.K.A.; data curation, H.S.Y.; writing—original draft preparation, F.K.A. and A.A.; writing—review and editing, S.D.; visualization, A.A.; supervision, F.K.A.; project administration, F.K.A. and M.K.; funding acquisition, F.K.A. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

This study was approved by the Standing Committee on Bioethical Research (SCBR), College of Pharmacy, Prince Sattam Bin Abdulaziz College, Saudi Arabia (research approval number SCBR-037-2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to university rules and regulations.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Schemes

Scheme 1. Graphical hypothesis.

Figure 1. Evaluation of the effect of caraway oil on CCI-induced paw cold allodynia using the acetone sprinkling test. It is represented as mean ± SEM; n = 6. a p ≤ 0.05 against sham control correspondent to the same day. b p ≤ 0.05 against CCI comparable to the same day. c p ≤ 0.05 versus caraway oil and anti-NGF in CCI compatible to the same day. * represents p-value ≤ 0.05.

Figure 2. Effect of Caraway oil on CCI-induced mechanical allodynia assessed by Von Frey hair test. It is represented as mean ± SEM; n = 6. a p ≤ 0.05 against sham control correspondent to the same day. b p ≤ 0.05 against CCI comparable to the same day. c p ≤ 0.05 versus caraway oil and anti-NGF in CCI compatible to the same day. * represents p-value ≤ 0.05.

Figure 3. Effect of caraway oil on CCI-induced mechanical hyperalgesia assessed by pinprick test. Values are given as mean ± SEM, n = 6. a p ≤ 0.05 versus sham control corresponding to same day. b p ≤ 0.05 versus CCI corresponding to same day. c p ≤ 0.05 versus Caraway oil and anti-NGF in CCI corresponding to same day. * represents p-value ≤ 0.05.

Figure 4. Effect of Caraway oil on CCI-induced heat hyperalgesia assessed by Eddy’s hot plate test. It is represented as mean ± SEM; n = 6. a p ≤ 0.05 against sham control correspondent to same day. b p ≤ 0.05 against CCI comparable to same day. c p ≤ 0.05 versus caraway oil and anti-NGF in CCI compatible to same day. * represents p-value ≤ 0.05.

Figure 5. Effect of caraway oil on CCI-induced rise in TNF-α level in the sciatic nerve. It is represented as mean ± SEM; n = 6. a p ≤ 0.05 against sham control correspondent to the same day. b p ≤ 0.05 against CCI comparable to same day. * represents p-value ≤ 0.05.

Figure 6. The effect of caraway oil on nerve growth factor and substance P in patients with CCI-induced neuropathy. It is represented as mean ± SEM; n = 6. a p ≤ 0.05 against sham control correspondent to same day. b p ≤ 0.05 against CCI comparable to same day. c p ≤ 0.05 versus caraway oil and anti-NGF in CCI compatible to same day. * represents p-value ≤ 0.05.

Scheme 2. Experimental design. A—Acetone sprinkling test; B—Pinprick test; C—Hot plate test; and D—Von Frey hair test.

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