New Treatment for the Cognitive and Emotional Deficits Linked with Paclitaxel-Induced Peripheral Neuropathy in Mice

Chemotherapy-provoked peripheral neuropathy and its linked comorbidities severely reduce the quality of a patient’s life. Its therapy is not completely resolved and has become an important clinical challenge. The protective actions of molecular hydrogen (H2) in many neurological disorders have been described, but its effects on memory and the emotional deficits accompanying neuropathic pain induced by chemotherapy remain unknown. In this study, using male mice injected with paclitaxel (PTX), we examined the effects of systemic treatment with hydrogen-rich water (HRW) in: (i) the mechanical and thermal allodynia provoked by PTX and the pathways involved; (ii) the memory deficits, anxiety- and depressive-like behaviors associated with PTX-induced peripheral neuropathy (PIPN); and (iii) the plasticity (p-extracellular signal-regulated protein kinase; p-ERK ½), nociceptive (p-protein kinase B, p-Akt), inflammatory (p-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; p-IKBα), and oxidative (4-hydroxynonenal: 4-HNE) alterations provoked by PIPN in the prefrontal cortex (PFC). The results revealed: (1) the antiallodynic actions of HRW administered at one or two times per day during 7 and 3 consecutive days; (2) the participation of Kv7 potassium channels and the Nrf2-heme oxygenase 1-NAD(P)H: quinone oxidoreductase 1 pathway in the painkiller effects of HRW; (3) the inhibition of memory deficits and the anxiodepressive-like behaviors related with PIPN induced by HRW; and (4) the normalization of p-ERK ½, p-Akt and 4-HNE up-regulation and the activation of antioxidant enzymes produced by this treatment in PFC. This study proposes HRW as a possible effective and safe therapy for PIPN and its associated cognitive and emotional deficits.


Introduction
Chemotherapy-provoked neuropathic pain is one of the prevalent adverse effects in cancer patients and an important clinical challenge because it severely reduces the patient's quality of life, and its therapy is not completely established [1,2]. Patients receiving chemotherapy also experience other symptoms, such as cognitive and affective disorders that can aggravate pain sensation and have a negative impact on the patient's well-being [3,4].
Currently, new chemotherapeutic agents with various mechanisms of action are continually being developed. However, despite these advances, classical agents such as paclitaxel (PTX) are also currently used to treat different types of cancer (lung, breast, ovarian, pancreas, melanoma) [5,6]. However, despite its clear clinical benefits, PTX-induced peripheral neuropathy (PIPN) [7,8] that is accompanied by memory deficits, depression, and anxiety, which in some cases can induce reduced treatment and/or even its discontinuation [9,10]. These symptoms have also been demonstrated in preclinical pain models [11][12][13][14]. At and the antioxidant enzymes HO-1, NQO1, glutathione S-transferase mu 1 (GSTM1) and superoxide dismutase 1 (SOD-1) in the PFC of mice with PIPN.

Animals
Male C57BL/6 mice (5-6-week-old and 21-25 g), acquired from Envigo Laboratories (Barcelona, Spain), were employed. The mice were maintained in regulated environmental conditions, 22 • C temperature, 66% humidity, and 12 h dark/light cycle, with food and water ad libitum. The experiments were started after 7 days to allow for the acclimatization of the housing conditions and performed between 9:00 a.m. and 5:00 p.m. The experiments were performed in agreement with the guidelines of the European Commission's directive (2010/63/EC) and the Spanish Law (RD 53/2013) regulating animal research, and the procedure was approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (ethical code 9863). Every effort was made to reduce the suffering and number of mice.

PTX Treatment
Painful neuropathy was provoked with the intraperitoneal administration of 2 mg/kg of PTX every other day, for four successive days, in accordance with previous studies [11,13]. PTX acquired from Tocris Bioscience (Bristol, UK) was dissolved in a 1:1:18 mixture solution containing Cremophor EL (Sigma-Aldrich, St. Louis, MO, USA), ethanol and saline (SS, 0.9% NaCl). Control groups were injected with vehicle solution in a similar schedule.

Allodynia Tests
Mechanical allodynia was assessed by determining the hind paw withdrawal response following the stimulation using von Frey filaments of distinct bending forces (North Coast Medical, Inc., San Jose, CA, USA). Animals remained inside the methacrylate cylinders (20 cm high per 9 cm diameter) (Servei Estació, Barcelona, Spain), placed in a grid bottom through the filaments were applied in the hind paws in agreement with the up-down paradigm [41]. The test started with a filament of 0.4 g, and the stiffness of the following filament was chosen according to the response. An Excel program (Microsoft Iberia SRL, Barcelona, Spain), which included curve fitting of the data, was used to calculate the threshold of the response. Shaking, withdrawing, or licking the paw was recorded as a positive reaction.
To evaluate cold allodynia, a cold plate analgesiometer (Ugo Basile, Italy) at 4 ± 0.5 • C was used, and the number of paw elevations was recorded for 5 min.
In both tests, both hind paws were tested.

Cognitive Behavior Test
The evaluation of the novel object recognition memory was performed in a gray box (44 × 44 cm) with four walls and a non-reflective base [42]. This test comprises four sessions of 10 min. On the first two days, the animals were habituated to the empty box. On day 3, the mice were put once again in the box and two equal objects were shown. At day 4, one of the familiar objects was changed to a new object. The time exploring the new and familiar objects was quantified. The discrimination index defined as ((time exploring the new object-time exploring the familiar object)/(time exploring the novel object + time exploring the familiar object) × 100) was utilized as a measure of cognitive behavior. A high discrimination index signifies good recognition memory.

Emotional Behavior Tests
The evaluation of depressive-like behaviors was performed by using the tail suspension test (TST) and the forced swimming test (FST). In both, the duration of immobility, as considered when the animals remained completely still, was quantified.
In the TST, the animals were suspended at 35 cm from the floor by using adhesive tape to the tip of the tail and fixing it to a surface. Their movements were filmed with a digital camera, and the immobility time was evaluated for 6 min [43].
In the FST, the mice were placed in methacrylate cylinders (25 cm high and 10 cm diameter) containing water, at 24 ± 2 • C, up to a 10 cm depth. Their activity was recorded with a digital camera for 6 min, and the time spent immobile during the last 4 min was registered [44].
The anxiety-like behaviors were assessed using the elevated plus maze (EPM) [45] and the open file (OF) tests [46].
The EPM apparatus used had 4 arms, two closed by 15 cm high walls and two open, elevated at 45 cm from the floor. Each of them was 5 cm wide and 35 cm long. The mice were always positioned in the center of the maze, facing the same open arm, and permitted to explore for 5 min. Their movements were filmed, and the number of entries into the closed and open arms, as well as the percentage of time passed in the open arms, were quantified.
In the OF test, the mice were positioned in the center of a box of 44 cm × 44 cm, enclosed by gray walls (30 cm high), and allowed to explore it for 5 min. They were filmed by a digital camera, and the number of entries and the percentage of time spent in the central area, as well as the number of squares crossed, were evaluated.
In all tests, the animals were familiarized with the testing space for 1 h prior to starting the proof, and these experiments were performed by researchers blinded to the experimental conditions.

Western Blot
At 21 days after PTX or vehicle injection, the animals were euthanized with cervical dislocation. PFCs were removed, frozen in dry ice, and maintained at −80 • C. The sonication of tissues was performed in a cold lysis buffer RIPA Buffer (Sigma-Aldrich, MO, USA). After solubilization for 1 h at 4 • C, the crude homogenates were sonicated again for 10 s and centrifuged at 700 g for 20 min at 4 • C. The supernatant (60 µg of total protein) was mixed with 4 x Laemmli loading buffer and loaded onto 4% stacking/12% separating sodium dodecyl sulfate-polyacrylamide gels. Next, the proteins were transferred onto a polyvinylidene fluoride membrane by electrophoresis (120 min) and blocked with phosphate-buffered saline (PBS) + 5% nonfat dry milk, PBS with Tween 20 + 5% bovine serum albumin (BSA), or Tris-buffered saline plus Tween 20 + 5% of nonfat dry milk or BSA, for 75 min. Then, the membranes were incubated with specific rabbit primary antibodies anti-p-ERK 1 /2 (1:250), ERK 1  After that, the blots were incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, UK), 1 h at room temperature, and the proteins were detected with chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed employing the Image-J program (National Institutes of Health, Bethesda, MD, USA).

Experimental Procedures
At first, in the PTX-and vehicle-injected mice, the mechanical and thermal antiallodynic effects induced by HRW or the vehicle administered at 1 time (1T) or 2 times (2T) per day during 7 consecutive days, from days 15 to 21 after PTX injection, were evaluated (n = 6 animals for each group). Considering the major analgesic effectiveness of HRW administered at 2T per day versus 1T per day, all of the following experiments were performed in animals treated with HRW at 2T per day.
The effects of HRW on memory deficits and depressive-and anxiety-like behaviors associated with PIPN were evaluated in different groups of vehicle-or PTX-injected mice treated with HRW, administered at 2T per day for 3 days (days 19 to 21 post-PTX injection). The behavioral tests were undertaken on the third day of HRW administration (21 days after PTX injection) (n = 8 animals for group).
All of the drugs were intraperitoneally administered to a final volume of 10 ml/kg. HRW at 0.3 mM was administered 1 h before testing, whereas 12 µmols/kg of XE-991, 25 mg/kg of ML-385, or 10 mg/kg of SnPP or dicoumarol were administered 30 min before the tests, in conformity with another study [27]. All of the drugs were newly prepared before administration. For each group treated with a drug, the respective control group received the same volume of analogous vehicles.

Statistical Analyses
The results are expressed as the mean values ± standard error of the mean (SEM). The three-way repeated measures ANOVA with time, treatment, and injection as the variable factors, followed by the one-way ANOVA and Student-Newman-Keuls tests, were applied to evaluate the effects of HRW administered alone or combined with XE-991, ML-385, SnPP, or dicoumarol on PTX-induced allodynia. The actions of the HRW treatment on the cognitive and emotive behaviors, as well as in the expression of several proteins, were analyzed by using a one-way ANOVA followed by the Student-Newman-Keuls test. The statistical analysis was performed utilizing the SPSS (version 28, IBM, Madrid, Spain) and Prism 8.0 (Graphpad, La Jolla, CA, USA) programs. A value of p < 0.05 was considered statistically significant.

The Mechanical and Cold Allodynia Provoked by PTX Were Inhibited by the Repetitive Administration of HRW
The three-way ANOVA repeated measures revealed significant effects of time, treatment, and injection and their interactions (p < 0.001) in both mechanical and thermal allodynia provoked by PTX in both paws.
In both hind paws, our results revealed that the mechanical allodynia provoked by PTX from day 14 to 21 after injection (p < 0.001; one-way ANOVA, against vehicle plus vehicle-treated animals; Figure 1A,C), was completely reversed by treatment with HRW administered at 1T and 2T per day during two consecutive days. The daily administration of HRW also progressively reduced the thermal allodynia induced by PTX injection (p < 0.001; one-way ANOVA, against vehicle plus vehicle-treated animals; Figure 1B,D), but five and three days of treatment with HRW, administered at 1T and 2T per day, were required for full inhibition. In both tests and paws, treatment with HRW administered at 1T or 2T per day did not produce any effect in the vehicle-injected animals.

Reversion of the Antiallodynic Actions Induced by HRW Treatment with a Kv7 Potassium Channels Blocker and Specific Nrf2, HO-1 and NQO1 Inhibitors
To explore the role of Kv7 potassium channels and the antioxidant pathway triggered by the Nrf2 transcription factor in the antiallodynic effects of HRW, the reversion of its effects with specific blockers or inhibitors was evaluated.
In both hind paws, a three-way repeated measures ANOVA showed significant effects for the time, treatment, and injection (p < 0.001), as well as the interactions between them (p < 0.001), in the reversion of the mechanical and thermal antiallodynic effects of HRW induced by the administration of XE-991 (a Kv7 potassium channel blocker), ML-385, SnPP, and dicoumarol (specific Nrf2, HO-1, and NQO1 inhibitors). That is, our results demonstrated that the repetitive administration of XE-991 (Figure 2A,B), ML-385 ( Figure 2C,D), SnPP ( Figure 3A,B), or dicoumarol ( Figure 3C,D) progressively reversed the mechanical and thermal antiallodynic effects of HRW. In all cases, while two days of treatment with the blocker or each inhibitor completely reversed the mechanical antiallodynic actions of HRW, the total reversion of the thermal antiallodynic effects of HRW required three days of treatment with XE-991, ML-385, SnPP, or dicoumarol (p < 0.001; one-way ANOVA, followed by the Student-Newman-Keuls test).  In all paradigms, treatment with XE-991, ML-385, SnPP, or dicumarol alone did not modify the mechanical and thermal allodynic responses provoked by PTX (Figures 2 and 3). Similar results were observed in the right hind paws of mice injected with vehicle-or PTX (results not displayed).

HRW Treatment Inhibited the Cognitive and Emotional Disorders Associated with PIPN
Our data confirmed the cognitive deficits ( Figure 4A) and depressive-like behaviors (Figure 4 B,C) associated with PIPN. Our results further revealed that the double administration of HRW per day, during 3 consecutive days, normalized the decreased discrimination index observed in PTX-injected animals in the novel object recognition test (p < 0.001; one-way ANOVA and Student-Newman-Keuls test; Figure 4A) and the augmented immobility time detected in PTX-injected mice in the TST (p < 0.001, one-way ANOVA and Student-Newman-Keuls test; Figure 4B) and FST (p < 0.001; one-way ANOVA and Student-Newman-Keuls test; Figure 4C). Thus, showing the recovery of memory deficits and the antidepressant effects produced by HRW treatment in animals with PIPN. The anxiety-like behaviors previously observed in PTX-injected mice were supported by the diminished number of entrances into the open arms (p < 0.001, one-way ANOVA test with respect to the vehicle plus vehicle-treated mice; Figure 5A) and the reduced time spent in the central area (p < 0.001, one-way ANOVA test with respect to the vehicle plus vehicle-treated mice; Figure 5D) of the EPM and OF tests. Our results further showed that the double daily administration of HRW normalized both diminished responses, thus revealing the anxiolytic effects of this treatment. The non-changes in the number of entries into the closed arms of the EPM (Figure 5B), nor in the number of squares crossed in the OF test ( Figure 5E) observed in the HRW-treated animals substantiated the absence of changes in the locomotor activity of these animals. Non-alterations in the percentage of time spent in the open arms of the EPM ( Figure 5C) nor in the number of entries in the central area of the OF ( Figure 5F) were detected.

Effects of Treatment with HRW on the Expression of p-ERK 1 /2, p-Akt, p-IKBα and BAX in the PFC of PTX-Injected Mice
Our findings demonstrated that PTX stimulated the phosphorylation of ERK 1 /2 (p < 0.017, one-way ANOVA and Student-Newman-Keuls test versus vehicle plus vehicletreated animals; Figure 6A), Akt (p < 0.008, one-way ANOVA and Student-Newman-Keuls vs. vehicle plus the vehicle-treated animals; Figure 6B), and IKBα (p < 0.010, one-way ANOVA and Student-Newman-Keuls vs. vehicle plus vehicle-treated animals; Figure 6D). The results further demonstrated that the HRW treatment normalized p-ERK 1 /2 and p-AKT overexpression but did not modify the PTX-induced p-IKBα upregulation. Thus, showing the inhibition of the plasticity and nociceptive changes induced by HRW treatment in the PFC of PTX-injected mice. Non-changes in the protein levels of BAX were observed ( Figure 6E). In all panels, * denotes significant changes vs. VEHI plus VEHI treated animals; + respect to VEHI plus HRW treated animals and $ respect to PTX plus HRW-treated mice (p < 0.05, one-way ANOVA and Student-Newman-Keuls test). Results are represented as mean values ± SEM; n = 8 animals per group.

Effects of Treatment with HRW on the Expression of 4-HNE, HO-1, GSTM1, NQO1 and SOD-1 in the PFC of PTX-Injected Mice
Considering the relevant participation of oxidative stress in the development of PIPN, the effects of treatment with HRW in the expression of 4-HNE and the antioxidant proteins HO-1, GSTM1, NQO1, and SOD-1 in the PFC of PTX-injected mice were evaluated.
Oxidative stress was confirmed by the increased expression of 4-HNE as compared with vehicle plus vehicle-treated mice (p < 0.001, one-way ANOVA and Student-Newman-Keuls; Figure 7A). Interestingly, treatment with HRW, in addition to normalizing 4-HNE overexpression, also augmented the levels of the antioxidant enzymes HO-1 (p < 0.011; ANOVA and Student-Newman-Keuls vs. vehicle plus vehicle and PTX plus vehicle-treated animals; Figure 7C) and GSTM1 (p < 0.010; ANOVA followed by Student-Newman-Keuls vs. vehicle plus vehicle and PTX plus vehicle-treated animals; Figure 7D). Thus, indicating the antioxidant properties of HRW in the PFC of PTX-injected animals. Figure 6. The effects of HRW on the p-ERK 1 /2, p-Akt, p-IKBα, and BAX expression in the PFC of PTX-injected animals. The protein levels of p-ERK 1 /2/ERK 1 /2 (A), p-Akt/Akt (B), p-IKBα/IKBα (D) and BAX (E) in the PFC of PTX-injected mice treated with HRW are shown. Control animals treated with vehicle (VEHI) plus VEHI are also displayed. Representative blots for p-ERK 1 /2, ERK 1 /2, p-Akt, and Akt (C), p-IKBα, IKBα, BAX and GAPDH (F) are presented. In all graphs, * denotes significant differences with respect to VEHI plus VEHI-treated mice and $ with respect to PTX plus HRW-treated mice (p < 0.05, one-way ANOVA and Student-Newman-Keuls test). Results are represented as mean values ± SEM (n = 3 samples per group).  In all panels, * indicates significant differences vs. the VEHI plus VEHI-treated mice, + indicates significant differences vs. the PTX plus VEHI-treated mice, and #, vs. PTX plus HRW-treated mice (p < 0.05, one-way ANOVA followed by the Student-Newman-Keuls test). Data are expressed as mean values ± SEM; n = 3 samples per group.
Regarding the protein levels of NQO1 ( Figure 7F) and SOD-1 ( Figure 7G), no significant changes were observed in PTX-injected mice treated with vehicle or HRW.

Discussion
This study demonstrated the inhibition of the allodynia, cognitive impairment and anxious-depressive-like behaviors induced by the repetitive treatment with HRW in mice with PIPN and revealed the likely pathways involved in these actions using pharmacological and molecular approaches.
Our results showed that the repetitive treatment with HRW, administered at 1T or 2T per day, reduced the mechanical and thermal allodynia provoked by PTX in both hind paws. These data evidenced, for the first time, the antiallodynic actions of HRW treatment in mice with PIPN. Our findings further showed that the double administration of HRW per day was more effective than the simple. Indeed, the PTX-induced allodynia was completely inhibited after three and seven days of treatment with HRW administered at 2T or 1T per day. These findings agree with the inhibitory actions produced by HRW or hydrogen-rich saline in mice with nerve injury-induced neuropathic pain [24][25][26][27], with the prevention of remifentanil-provoked post-surgical hyperalgesia [50] and with the improved hyperalgesia induced by oxaliplatin [51]. Our results also supported the major effectiveness produced by the double daily administration of HRW versus the simple as recently demonstrated in animals with neuropathic pain provoked by nerve injury [27].
Regarding the main pathways involved in the analgesic actions of HRW in animals with PIPN, we evaluated the possible participation of Kv7 potassium channels by assessing the reversion of HRW effects with the co-treatment with the selective Kv7 potassium channels blocker, XE-991. Our results showed that the inhibition of the mechanical and thermal allodynia produced by HRW was reversed with the administration of XE-991. These data agreed with the reversion of the antinociceptive effects produced by several H 2 S donors with the administration of Kv7 potassium channels blockers during chronic pain [31] and further revealed the participation of these channels in the pain-relieving actions of HRW in mice with PIPN.
Oher works showed the crucial role played by the Nrf2 transcription factor in the effects produced by H 2 in different experimental conditions [18,52]. In this study, we assessed the importance of this pathway in the painkilling actions of HRW in mice with PIPN. The reversal of the analgesic effects of HRW with the co-administration with ML-385, SnPP, or dicoumarol revealed the involvement of the antioxidant system in the inhibition of the mechanical and thermal allodynia produced by HRW in animals with PIPN. These data supported other studies made with H 2 [27] or H 2 S donors, which also activated the Nrf2/HO-1 and/or NQO1 path for inhibiting nerve injury-induced neuropathic pain in rodents [33].
This work confirmed the memory deficits caused by PTX, as previously demonstrated by the reduced discrimination index in the novel object recognition test [14], and further proved that the administration of HRW normalized this cognitive impairment. These results are in accordance with the prevention of stress-induced decline of memory produced by the continuous consumption of HRW in mice [53], with the efficacy of other gases in inhibiting the memory deficits associated with chronic osteoarthritis pain [54] as well as with those produced by 7-chloro-4-(phenylselanyl) quinoline in animals with PIPN [55]. Several works indicated the contribution of the plasticity changes in memory impairments and that several drugs can improve these deficits by restoring the altered expression of MAPK activated by chemotherapy in the CNS [14]. The reversion of the increased expression of p-ERK 1 /2 caused by PTX with the HRW treatment agreed with the effects produced by this treatment in the PFC of sciatic nerve-injured mice [27] and further suggested that the normalization of the plasticity changes produced by HRW might be involved in the recovery of memory deficits produced by this treatment in PTX-injected mice.
In accordance with a recent publication [27], our data revealed the antidepressant effects of HRW in animals with PIPN. In compliance, another work showed that HRW could prevent chronic stress-induced depressive-like behaviors in mice [21]. Even so, our results showed, for the first time, the therapeutic potential of HRW against the depressive-like behaviors associated with PIPN. Moreover, considering that classic antidepressants produce a wide range of side effects and that treatment with HRW is considered a safe treatment with non-demonstrable relevant secondary effects [18], the therapeutic administration of HRW might be considered an effective alternative for treating the depressive-like behaviors linked with PIPN.
Our data further confirmed the anxiolytic-like behaviors related to PIPN [11,13] and revealed the anxiolytic properties of treatment with HRW in this pain model. Indeed, HRW reversed the low number of entries into the open arms and the low time passed in the central area of the EPM and OF tests, as displayed by PTX-injected mice treated with vehicles. The non-alterations in the locomotor activity induced by HRW were demonstrated by the absence of changes in the number of entries into the closed arms and in the number of squares traversed in the EPM and OF tests, respectively. Thus, indicating that the anxiolytic actions of HRW were not affected by possible locomotory alterations. These data concur with the anxiolytic properties of HRW in animals with neuropathic pain generated by nerve injury [27].
One of the multiple mechanisms underlying the affective disorders associated with PIPN is oxidative stress [13,56]. Moreover, the antidepressant and anxiolytic actions induced by several compounds are mainly produced by inhibiting oxidative stress [13,27,33]. In accordance, the oxidative-stress responses induced by PTX demonstrated by the increased expression of 4-HNE in PFC [13] and its normalization with HRW treatment revealed the antioxidative effects of H 2 in the CNS of mice with PIPN. Accordingly, HRW also reversed the oxidative stress caused by oxaliplatin [51]. Our data also revealed an improvement in the HO-1 and GSTM1 expression in the PFC of HRW-treated animals as occurs with CoPP treatment [13]. Moreover, considering the antidepressant and anxiolytic properties of several antioxidant compounds, our results suggested that the antioxidant actions of HRW might be involved in the inhibition of the emotional disorders linked with PIPN. Nevertheless, because p-ERK 1 /2 also takes part in the manifestation of the anxiodepressive-like behaviors by triggering inflammatory and oxidative responses in the cortex, and its inhibition avoided the affective alterations accompanying chronic pain [57], the stabilization of p-ERK 1 /2 upregulation produced by HRW in PFC might also be engaged in the inhibition of the anxious-depressive-like behaviors linked with PIPN. Lastly, the non-changes in BAX expression observed in the PFC of animals injected with PTX alone or combined with HRW revealed that the apoptotic responses provoked by this chemotherapeutic agent in other brain areas, such as the hippocampus [58], are not observed in PFC [13].
In recent years, several works have investigated different treatments for preventing or treating chemotherapy-induced neuropathic pain, such as the administration of channel and neurotransmitter modulators, neuroprotectors, antidepressants, anticonvulsants, or opioids [59]. However, most of these therapies exhibited moderate efficacy and significant side effects such as somnolence, constipation, tolerance, and dizziness [60]. In addition, some treatments only improved neuropathy or one of the emotional disorders associated with chemotherapy-induced neuropathic pain [61][62][63], but only a few studies evaluated the possible effects of different treatments in the management of PIPN-associated mood disorders [13,64,65]. In this line, this study demonstrated that treatment with HRW inhibited neuropathy and avoided the cognitive and emotional deficits associated. Moreover, because the main nociceptive, cognitive, and emotional symptoms observed in our animal model are very similar to those observed in patients receiving chemotherapy [9], considering the few side effects induced by HRW, this treatment might be proposed as a safe and effective therapy against PIPN and the comorbidities associated.

Conclusions
In summary, this research demonstrates that the repetitive administration of HRW inhibited allodynia, memory deficits, and anxious-depressive-like behaviors provoked by PTX. This treatment normalized the plasticity changes, nociceptive responses and oxidative stress provoked by PTX and improved the expression of antioxidant enzymes, HO-1 and GSTM1, in the PFC of mice with PIPN. In summary, this work suggests that HRW treatment might be a good approach for the management of PIPN and its accompanying cognitive and emotive disorders.