The antihyperalgesic effect of docosahexaenoic acid in streptozotocin-induced neuropathic pain in the rat involves the opioidergic system

Author’s Accepted Manuscript The antihyperalgesic effect of docosahexaenoic acid in streptozotocin-induced neuropathic pain in the rat involves the opioidergic system Arizai Yolia Landa-Juárez, Francisca PérezSeveriano, Gilberto Castañeda-Hernández, Mario I. Ortiz, Aracely Evangelina Chávez-Piña www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(18)30751-9 https://doi.org/10.1016/j.ejphar.2018.12.029 EJP72134

To appear in: European Journal of Pharmacology Received date: 6 July 2018 Revised date: 6 December 2018 Accepted date: 20 December 2018 Cite this article as: Arizai Yolia Landa-Juárez, Francisca Pérez-Severiano, Gilberto Castañeda-Hernández, Mario I. Ortiz and Aracely Evangelina ChávezPiña, The antihyperalgesic effect of docosahexaenoic acid in streptozotocininduced neuropathic pain in the rat involves the opioidergic system, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.12.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The antihyperalgesic effect of docosahexaenoic acid in streptozotocininduced neuropathic pain in the rat involves the opioidergic system

Arizai Yolia Landa-Juáreza, Francisca Pérez-Severianob, Gilberto CastañedaHernándezc, Mario I. Ortizd, Aracely Evangelina Chávez-Piñaa* a

Laboratorio de Farmacología, Doctorado en Biotecnología, Escuela Nacional de

Medicina y Homeopatía, Instituto Politécnico Nacional, Ciudad de México, México. b

Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía

"Manuel Velasco Suárez", Ciudad de México, México. c

Departamento de Farmacología, Centro de Investigación y de Estudios

Avanzados del Instituto Politécnico Nacional, Ciudad de México, México. d

Área Académica de Medicina, del Instituto de Ciencias de la Salud, de la

Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo, México. *

Corresponding autor: Escuela Nacional de Medicina y Homeopatía del Instituto

Politécnico Nacional, Guillermo Massieu Helguera No. 239, Fraccionamiento “La Escalera”, Ticomán, México, D.F. C.P. 07320, Mexico; Tel.: + 52 57296000 x 55583; [email protected].

Abstract Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that has shown an antinociceptive effect in multiple pain models, such as inflammatory and

1

neuropathic pain by chronic constriction injury in rats; however, its mechanism of action is still not well-understood. Reports suggest that DHA activates opioid signaling, but there is no information on this from a model of neuropathic pain. As a result, the aims of this study were (1) to determine the antihyperalgesic and antiallodynic effect of peripheral DHA administration, and (2) to evaluate the participation of the opioid receptors in the antihyperalgesic effect of DHA on streptozotocin-induced neuropathic pain in the rat. Female Wistar rats were injected with streptozotocin (50 mg/kg, i.p.) to induce hyperglycemia. The formalin, Hargreaves, and von Frey filaments tests were used to assess the nociceptive activity. Intraplantar administration of DHA (100–1000 μg/paw) or gabapentin (5621778 μg/paw) decreased formalin-evoked hyperalgesia in diabetic rats, in a dosedependent manner. Furthermore, DHA (562 μg/paw) and gabapentin (1000 μg/paw) reduced thermal hyperalgesia and allodynia. Local peripheral administration of naloxone (non-selective opioid receptor antagonist; 100 μg/paw), naltrindole (selective δ receptor antagonist; 1 μg/paw), and CTOP (D-Phe-Cys-TyrD-Trp-Orn-Thr-Pen-Thr-NH2, μ receptor antagonist; 20 μg/paw) prevented formalin-evoked hyperalgesia in diabetic rats but not by GNTI (guanidinonaltrindole, κ receptor antagonist;1 µg/paw). It is suggested that peripheral DHA shows an antihyperalgesic effect in neuropathic pain in the rat. Furthermore, δ and μ receptors are involved in the antihyperalgesic peripheral effect of DHA in diabetic rats. Keywords: Docosahexaenoic acid; neuropathic pain; streptozotocin; opioid; allodynia; hyperalgesia.

2

1. Introduction Neuropathic pain is defined for the International Association for the Study of Pain (IASP) as “pain caused by a lesion or disease of the somatosensory nervous system either in the periphery or centrally” (Jensen et al., 2011; Woolf, 2004). Diabetic neuropathy affects 347 million people around the world; the most common symptoms in neuropathy due to nerve injury are spontaneous pain, tactile allodynia (pain response to innocuous stimulus), and hyperalgesia (pain amplified response to noxious stimulus) (Sambasevam et al., 2017; Woolf, 2004). Pharmacological treatment for neuropathic pain is limited because more than two-thirds of patients do not obtain satisfactory pain relief (Jensen et al., 2011). The first line for the treatment of neuropathic pain are anticonvulsants, antidepressants, and opioid analgesics. However, those induce adverse effects, such as drowsiness, constipation, cognitive impairment, and addiction (Baron et al., 2010; Sambasevam et al., 2017). Docosahexaenoic acid (DHA, 22:6 n-3), an omega-3 (ω-3) long-chain polyunsaturated fatty acid has shown anti-inflammatory, antinociceptive, antioxidative, gastroprotective and neuroprotective effects in experimental murine models (Bento et al., 2011; Nakamoto et al., 2010; Pineda-Peña et al., 2018; Türkez et al., 2012). Furthermore, DHA’s antinociceptive effect after oral administration has been reported against thermal and chemical stimulus in mice (Nakamoto et al., 2010); recently our group demonstrated its oral and local antinociceptive effect on the rat formalin test (Arroyo-Lira et al., 2014; 2017; LandaJuárez et al., 2016). A recent study suggests that DHA activates opioid signaling

3

modulating the synthesis of opioid peptides in models of Complete Freund’s adjuvant-induced chronic pain and acetic acid-induced pain (Torres-Guzman et al., 2014; Nakamoto et al., 2011). Moreover, DHA has shown an antiallodynic effect after chronic constriction injury-induced neuropathic pain (Huang and Tsai, 2016; Manzhulo et al., 2015) and antihyperalgesic effect in a model of partial sciatic nerve ligation (Silva et al., 2017). Pretreatment with systemic DHA prevented thermal hyperalgesia and mechanical allodynia in a streptozotocin-induced neuropathic pain model (Heng et al., 2015). In the same study, it was evidence that DHA supplementation decreased the excitability of dorsal root ganglion neurons by decreasing sodium and increasing potassium currents. However, the antihyperalgesic effect of DHA in streptozotocin-induced neuropathic pain has not been well understood. The objectives of this study were: (1) to determine the anti-hyperalgesic and anti-allodynic effect of peripheral DHA administration and (2) to evaluate the participation of the opioid receptors in the antihyperalgesic effect of DHA on streptozotocin-induced neuropathic pain in rats.

2. Material and methods 2.1. Animals Female Wistar rats (7–9 weeks: weight range 180–220 g) from our own breeding facilities were used in this study. Rats were housed in an animal room at 22 ± 2°C with a 12:12 light-dark cycle and food and drinking water ad libitum before the 4

experiments. The study was conducted according to the ethical guidelines on ethical standards for the investigation of experimental pain in animals (Zimmermann, 1983) and the protocol was approved by an independent committee at our institution (Protocol ENMH-CB-144-2015). Efforts were made to minimize animal suffering and to reduce the number of animals used, which were used only once. At the end of the experiments, rats were euthanized with intramuscular administration of ketamine and xylazine (60 mg/kg and 30 mg/kg, respectively).

2.2. Drugs Docosahexaenoic acid (DHA), olive oil, streptozotocin, 5'-guanidinonaltrindole (GNTI), and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) were purchased from Sigma-Aldrich (Toluca, Mexico). Ketamine and xylazine were obtained from PISA (Mexico). Gabapentin was purchased with Cayman Chemical (Michigan, USA), naltrindole and naloxone hydrochloride were purchased from Santa Cruz Biotechnology (Mexico). Formaldehyde was purchased from J.T. Baker (Mexico). All drugs were dissolved in isotonic saline solution, except streptozotocin, which was dissolved in injectable water. The vehicle used for DHA was olive oil. All drugs were injected in a volume of 50 μl per paw.

2.3. Induction to experimental diabetes mellitus by streptozotocin Rats were treated with streptozotocin (50 mg/kg; i.p.) to produce experimental diabetes mellitus type 1 (Ma et al., 2015). Control animals received injectable water. Diabetes mellitus was confirmed 48 h after streptozotocin injection and 24 h 5

before each experiment by measurement the tail vein blood glucose levels using a Glucometer OneTouch® UltraMini (Roche, Mexico). Only animals with a final blood glucose level greater than 250 mg/dl were considered as diabetic and included in the study.

2.4. Assessment of formalin-induced flinching hyperalgesia in rats The 1% formalin test in the paw was used to assess nociception and antinociception using the previously described formalin test (Arroyo-Lira et al., 2017; Ortiz, 2012). Briefly, streptozotocin-induced hyperglycemic rats (after 4-5 weeks) were injected with fifty microliters of diluted formalin (1%) subcutaneously (s.c.) into the plantar surface of the right hind paw, and the incidence of spontaneous flinching behavior was quantified for 1 min every 5 min for a period of 60 min after the injection. The data collected between 0 and 10 min post-formalin injection represent phase 1, and the data collected between 15 and 60 min represent phase 2. The person performing the experiments was unaware of the treatments that the rats received. For all the experiments, the drug doses and administration time schedules used were selected based on previous reports (Landa-Juárez et al., 2016; Torres-López et al., 2007). A previous study evaluated the vehicle group of formalin and found that a typical nociceptive response pattern is not generated. Therefore, this group was not performed in our experiments (Quiñonez-Bastidas et al., 2018). The areas under the resulting curves (AUC) were calculated using the trapezoidal rule. The AUC was calculated for the two phases of the assay, and the percent of antinociception for each phase was calculated according to the following equation (Ortiz and Castañeda-Hernández, 2008): 6

Percent of antinociception= [(AUC vehicle - AUC post-compound) / AUC vehicle] x 100

2.5. Measurement of thermal hyperalgesia activity Thermal hyperalgesia to radiant heat was assessed using the Hargreaves test in streptozotocin-induced diabetic rats (after 4-5 weeks) (Hargreaves et al., 1988). Tests were conducted at room temperature (~25°C). Briefly, rats were placed in a Plexiglas chamber on an elevated clear platform on a 336 Plantar Test Apparatus (IITC Life Science). Following 30 min acclimatization period, a radiant heat source with infrared radiation was applied through the glass to the plantar surface of the hind paw. The paw withdrawal latency (PWL), defined as the time from onset of the radiant heat to the withdrawal of the paw, was determined using a focused beam of light with an active intensity of 26% maximal, a cutoff of 20 s, and idle intensity of 5% maximal. 100% beam intensity heats to ~250°C. To measure paw withdrawal latency, a timer was automatically initiated at the onset of heat application (active intensity), and upon paw withdrawal, the heat source was returned to idle intensity automatically stopping the timer. Three independent measurements each hour, separated by 5 min, for 6 h, were used to determine paw withdrawal latency (O´Brien et al., 2015; Ortiz et al., 2007). Due to their different sensitivities in the strain of the experimental animals, the Hargreaves experiment was carried out at different active intensities (20–50%) of the radiant focus to obtain the appropriate intensity that generates a latency of the extraction of the paw between 9 and 12 s (Morales et al., 2009), and based on our experiments, an active intensity of 26% was determined for the evaluation of thermal hyperalgesia. 7

The areas under the PWLs against time curves (AUC) were calculated by the trapezoidal rule. AUC was calculated, and percent antihyperalgesia was calculated according to the following equation (Ortiz et al., 2007): Percent of antihyperalgesia = [(AUCpost compound – AUCvehicle)/AUCvehicle] x 100

2.6. Measurement of tactile allodynia In the present study, tactile allodynia was evaluated 6 to 14 weeks after streptozotocin-induced diabetic rats. Allodynia was developed in the rats until the 14th week of the evolution of streptozotocin-induced diabetic rats. Animals were acclimatized to cages with wire mesh bottoms for 30 min. A series for von Frey filaments (Touch-Test Sensory Evaluator [EXACTATM]) of 0.4, 0.6, 1, 2, 4, 6, 8, and 15 g were used to determine the 50% paw withdrawal threshold using the up-down method of Dixon (1980). A series of filaments starting with a buckling weight of 2 g, was applied in consecutive sequence to the plantar surface of the right hind paw for 5 s with pressure causing the filament to buckle. The lifting of the paw was recorded as a positive response and prompted the use of the next lighter filament, whereas the absence of paw withdrawal after 5 s indicated a negative response and prompted the use of the next filament of increasing weight. This procedure continued until four more measurements have been made after the initial change of the behavioral response or until five consecutive negative (assigned a score of 15 g) or four consecutive positive (assigned a score of 0.25 g) responses had occurred. The resulting scores were used to calculate the 50% response threshold using the formula: 50% g threshold = (10[Xf + κδ]) /10000, where Xf =the value (in log units) of the final von Frey filament used, κ = tabular value 8

(from table in [Chaplan et al., 1994]) for the pattern of positive and/or negative responses, and δ = mean difference (in log units) between stimuli (here 0.22486). Allodynia was present when paw withdrawal thresholds were 4 g or less (AraizaSaldaña et al., 2015).

2.7. Study design On the formalin test, rats received vehicles or increasing doses of DHA (100, 300, 562 and 1000 μg/paw, 75 min before formalin insult) or gabapentin (562, 1000 and 1778 μg/paw, 60 min before formalin insult) into the right paw to determine whether these drugs acted locally. To assess if the antihyperalgesic effect of drugs was due to a local action, formalin was administered in the right paw and the test drugs [DHA (562 μg/paw, 75 min before formalin insult) or gabapentin (1778 μg/paw, 60 min before formalin insult)] in the contralateral paw (s.c.). Based on our results, vehicles, DHA (562 μg/paw, 75 min before the tests) or gabapentin (1778 μg/paw, 60 min before the tests) were administered individually to the right (ipsilateral) or left (contralateral) paws before the performance of thermal hyperalgesia and tactile allodynia assays. To assess whether the antihyperalgesic effect induced by DHA in the formalin test is induced by the local peripheral participation of the opioidergic system, the non-selective opioid receptor antagonist naloxone (100 μg/paw) was injected 20 min before DHA (562 μg/paw; 75 min before formalin insult). Furthermore, to define the subtype of the opioid receptor involved in the antihyperalgesic effect of DHA in the formalin test, the selective δ receptor, δ antagonist naltrindol (1 μg/paw), and the κ receptor, κ antagonist GNTI (1 µg/paw), 9

were administrated 20 min before DHA, or the μ receptor, μ antagonist CTOP (20 μg/paw), was administered 45 min before DHA or their vehicles (NaCl 0.9%) in the formalin test. Doses and times of opioidergic antagonists were selected based on previous reports (Izquierdo et al., 2013; Chung et al., 2012; Brigatte et al., 2013).

2.8. Statistical analysis All experimental results are given as the mean ± S.E.M. of the data obtained from 6–12 animals per group. Differences between the groups were determined by the Student's test, one-way analysis of variance (ANOVA), followed by Newman–Keuls post-hoc test or two-way analysis of variance (ANOVA), followed by Bonferroni’s post-hoc test. Differences were considered to reach statistical significance when the P-value was less than 0.05. All statistical analyses were conducted using Graph Pad Prism 5 software.

3. Results 3.1. Induction to experimental diabetes by streptozotocin Rats injected with streptozotocin showed a decrease (P < 0.05) in body weight compared with controls. Streptozotocin, but not water injection, caused hyperglycemia. The blood glucose levels in these rats were greater than 250 mg/dl 48 h after streptozotocin injection (P < 0.05). Over the following 4 and 14 weeks of the experiments, the blood glucose levels were maintained in streptozotocininjected rats while control rats were normoglycemic (Table 1). In addition to the loss of weight and hyperglycemia, diabetic rats also showed an increase in food 10

intake (polyphagia), water intake (polydipsia), and excreted urine volume (polyuria) (data not shown).

3.2. Effect of docosahexaenoic acid on formalin-evoked hyperalgesia Both the non-diabetic and diabetic groups exposed to subcutaneous injection of formalin produced a biphasic pattern on flinching. Phase 1 of the nociceptive response began immediately after formalin administration and then declined within approximately 10 min, and phase 2 began 15 min after formalin administration and lasted about 1 h (data not shown). Compared to non-diabetic rats, peripheral administration of formalin in diabetic rats produced an increase in flinching behavior indicative of hyperalgesia, as shown in the AUC of the number of flinches of the paw of the rats on phase 2 but not in phase 1 of the formalin test (P < 0.05; Fig. 1). Ipsilateral local peripheral administration of DHA produced a dosedependent antihyperalgesic effect on formalin phase 2 (Fig. 2). Contralateral peripheral administration of DHA (562 µg/paw) did not produce a significant antihyperalgesic effect (Fig. 2). The peripheral administration of gabapentin in the ipsilateral paw produced a dose-dependent antihyperalgesic effect on formalin phase 2; while gabapentin (1778 µg/paw) in the contralateral paw exhibited a significant antihyperalgesic effect (Fig. 2). We can deduce that the effect of DHA was attributed to local peripheral effects because there is no observed antihyperalgesic effect after administration into the contralateral paw. Based on our results, we selected the dose in which compounds reached their maximal effect (562 µg/paw of DHA and 1000 µg/paw of gabapentin). DHA or gabapentin did not induce an antihyperalgesic effect on formalin phase 1 (data not shown). 11

3.3. Effect of docosahexaenoic acid on thermal hyperalgesia In diabetic rats, the baseline latencies to heat-induced pain reaction were decreased compared to non-diabetic rats, confirming the development of thermal hyperalgesia (Fig. 3). Peripheral administration of DHA or gabapentin exhibited an antihyperalgesic effect. DHA did not induce an antihyperalgesic effect administered in the contralateral paw, while gabapentin administered in the contralateral paw exhibited the same percentage of thermal antihyperalgesia than gabapentin administered in the ipsilateral paw, demonstrating that thermal antihyperalgesic effect of DHA is local and not systemic (Fig. 4).

3.4. Tactile allodynia After 14 weeks, when comparing hyperglycemic and non-diabetic rats, reduced below 4 g the 50% paw withdrawal threshold response in the ipsilateral paw, which is indicative of tactile allodynia induction. Ipsilateral administration of DHA and gabapentin significantly attenuated tactile allodynia during 4 h of the experiment. However, the paw withdrawal threshold was reduced at the end of the experiment (6 h). Contralateral DHA or gabapentin administration did not show an antiallodynic effect, demonstrating that the anti-allodynic effect of DHA is local and not systemic (Fig. 5).

3.5. Antagonism of opioid receptors on the anti-hyperalgesic effect of DHA on the formalin test

12

Naloxone (non-selective receptor opioid antagonist), naltrindole (selective δ receptor antagonist) or CTOP (selective µ receptor antagonist), but not vehicles, significantly reversed the antihyperalgesic effect of DHA on formalin test (Fig. 6, 7, and 8). In contrast, GNTI (selective κ receptor antagonist) was unable to prevent the DHA-induced antihyperalgesic effect on diabetic rats (Fig. 9). Opioidergic antagonists alone did not significantly alter the hyperalgesic effect induced by formalin in diabetic rats. Local peripheral treatment with DHA, opioidergic antagonists or a mixture of these compounds did not alter the ambulation or motor capacity in the experimental animals (data not shown).

4. Discussion This study pharmacologically demonstrates that local peripheral administration of DHA reverted hyperalgesia and allodynia in neuropathic pain in streptozotocininduced diabetic rats, µ and δ receptors are involved in the antihyperalgesic effect of DHA in neuropathic pain induced in diabetic rats. This effect is likely due to a purely local peripheral action, as DHA administration in the contralateral paw fails to produce antinociception in the formalin, Hargreaves, and von-Frey tests. This allows us to disregard the systemic effect. On the other hand, peripheral administration of gabapentin on the contralateral paw was able to produce antihyperalgesia in all of the tests. Therefore, DHA exhibits a safer profile than gabapentin.

13

Diabetic peripheral neuropathy is one of the major complications caused by long-standing hyperglycemia, and the most common symptoms are hyperalgesia and allodynia as it was observed in our experiments (Kishore et al., 2018; Sambasevam et al., 2017; Woolf, 2004). Several studies have used streptozotocin for induction of diabetes to develop neuropathic pain in animals where hyperalgesia usually is developed at 4 weeks (Zhao et al., 2014) and allodynia at 6 weeks after streptozotocin injection (Rocha-González et al., 2014). In our study, hyperalgesia was developed in accordance with previous reports (4–5 weeks) and confirmed in the formalin and thermal hyperalgesia models; in contrast, in our experiments allodynia was performed until 14 weeks after streptozotocin injection; several authors have reported that allodynia was developed after 6 weeks of streptozotocin injection. However, allodynia is not always developed in a determined time, for example, there are other reports that after 6, 8 or 12 weeks of streptozotocin injection allodynia is presented (Araiza-Saldaña et al., 2015; RochaGonzález et al., 2014; Yamamoto et al., 2009). Neuropathic pain is an important public health problem, and the currently used drugs have limited efficacy and increasing adverse effects. Consequently, there is a significant need for further development of novel medications based on its molecular target for its treatment (Salat et al., 2018; Sambasevam et al., 2017). This is the first report showing that local DHA induces antihyperalgesic and antiallodynic effects in models of neuropathic pain in diabetic rats. Omega-3 polyunsaturated fatty acids are essential nutrients for humans, and are involved in a variety of biological functions such as neuroprotective (Mayurasakorn et al., 2011), antioxidative (Türkez et al., 2012), and anti-inflammatory effects (Bento et 14

al., 2011). Previously, it has been reported that oral (Arroyo-Lira et al., 2014; 2017; Nakamoto et al., 2010), intracerebroventricular (Nakamoto et al., 2011), and local peripheral administration of DHA exhibits an antinociceptive effect in the formalin nociceptive tests with normoglycemic animals (Landa-Juárez et al., 2016). However, there are no reports of DHA’s antihyperalgesic effect in diabetic neuropathy. Previous reports have shown that DHA supplementation inhibited neuropathic pain in rats in a model of chronic constriction injury (Heng et al., 2015; Huang and Tsai, 2016; Manzhulo et al., 2015). Also, Redivo et al. reported that treatment with fish oil prevented mechanical allodynia in diabetic rats (Redivo et al., 2016). Interestingly, a case series study reported that five patients with several types of neuropathic pain and subjected to daily treatment with fish oil had a reduction in the clinical score of pain, without significant adverse effects (Ko et al., 2010). Nevertheless, the precise mechanism underlying the effect of DHA relieving neuropathic pain is poorly understood. Most of the information about the antinociceptive mechanism of DHA comes from studies addressing inflammatory pain. For example, the antinociceptive effect of DHA has been related to the activation of big- and small-conductance Ca2+-activated K+ channels (KCa1.1, KCa2.1-3) and ATP-sensitive K+ channels (Kir6.1-2) on the 1% rat formalin test in non-diabetic rats (Landa-Juárez et al., 2016). Furthermore, many polyunsaturated fatty acids and their metabolites have been demonstrated to directly bind and modulate transient receptor potential ion channels (TRP). Resolvin D1 (RvD1), a DHA metabolite, induces inhibitory actions on TRPA1 (transient receptor potential ankyrin), TRPV3, and TRPV4 (transient receptor potential vanilloid) (Bang et al., 15

2010; 2012). Besides, in the inflamed tissue of pain models, resolvin E1, an eicosapentaenoic acid metabolite, reduced expression of proinflammatory cytokines and chemokines, such as IL-1β, IL-6, and TNF-α (Xu et al., 2010). However, there is no information about the anti-hyperalgesic mechanism of DHA in models of neuropathic pain in diabetic rats. Our study also reveals, for the first time, that the anti-hyperalgesic effect of DHA on streptozotocin-induced neuropathic pain is dependent on the modulation of the opioidergic system. Attenuation of the antihyperalgesic effect of DHA with local pretreatment of naloxone, CTOP, and naltrindole confirm the participation of peripheral μ and δ receptors. Recently, it has been published that the antinociceptive effect of DHA is mediated by β-endorphin (Nakamoto et al., 2011). Then, DHA might produce antinociception on neuropathic pain in diabetic rats via the release of several endogenous opioid peptides, such as β-endorphin and met-enkephalin, because those endogenous opioids possess high affinity for μ and δ receptors (Kapitzke et al., 2005). Furthermore, it was confirmed that β-endorphin itself also produced an antinociceptive effect and that its effect was mediated by μ and δ receptors, but not by κ receptors, in a model of inflammatory pain such as the acetic acid-induced writhing test (Nakamoto et al., 2011). Based on the finding that the peripheral opioid receptors are involved in the DHA antihyperalgesia effect, it could be interpreted that DHA acts indirectly, by an unknown pathway, in μ and δ receptors influencing the endogenous opioidergic system to produce antinociception in diabetic rats. In addition, other compounds have shown indirect activation of opiodergic system in their antinociceptive effect, such as the epoxyeicosapentaenoic acid, another metabolite of DHA via cytochrome P450 16

enzyme (Wagner et al., 2017), curcumin (Banafshe et al., 2014), diosmin (Carballo-Villalobos et al., 2018), cardamonin (Sambasevam et al., 2017), and 7hydroxy-3,4-dihydrocadalin (Rocha-González et al., 2014) due to their antinociceptive effect was blocked by naloxone in a model of neuropathic pain. Only a few reports have studied the possible mechanism of the antinociceptive effect of DHA in neuropathic pain. DHA supplementation decreased the excitability of dorsal root ganglion (DRG) neurons by decreasing the sodium currents and increasing potassium currents in diabetic rats (Heng et al., 2015). It is well-known that activation of potassium channels is modulated by several analgesic drugs, such as opioids, then those mechanisms might be related to the antihyperalgesic effect of DHA in neuropathic pain, as potassium channel activation was described earlier in the antinociceptive effect of DHA in a model of inflammatory pain (LandaJuárez et al., 2016; Ocaña et al., 1993; Ortiz et al., 2005; 2007). Besides, fish oil supplementation reduced TNF-α in the spinal cord and decreased sciatic myeloperoxidase activity in a model of neuropathic pain induced by sciatic nerve ligation (Silva et al., 2017). RvD1 exhibits regulation of inflammatory mediators and the inhibition of NF-κB/p65 and p-ERK pathways in a non-compressive lumbar disk herniation model (Liu et al., 2016). Thus, there is still a lack of the antineuropathic mechanism of DHA that needs to be further investigated.

5. Conclusions Our study suggests that local peripheral administration of DHA reverted hyperalgesia and allodynia in diabetic rats. Furthermore, the results seem to

17

indicate that the antihyperalgesic effect of DHA involves the indirect activation of µ and δ receptors.

Conflict of Interest Statement The authors report no conflict of interest

Acknowledgments The authors acknowledge the support provided by the National Council for Science and Technology (Project CONACyT 178027) and SIP 20180451. Arizai Yolia Landa Juárez is a CONACyT fellow (Grant Number 488726). Authors acknowledge to Q.F.B. Martha Patricia González García from Pharmacology DeparmentCinvestav, M.V.Z. Ricardo Gaxiola Centeno, M.V.Z. Rafael Leyva Muñoz, M.V.Z. Benjamín Chávez Álvarez, M.V.Z. Carlos Giovanni Sam Miranda and René Pánfilo Morales from Cinvestav-UPEAL for their technical assistance in this project. References Araiza-Saldaña, C.I., Pedraza-Priego, E.F., Torres-López, J.E., Rocha-González, H.I., Castañeda-Corral, G., Hong-Chong, E., Granados-Soto, V., 2015. Fosinopril prevents the development of tactile allodynia in a streptozotocininduced diabetic rat model. Drug. Dev. Res. 76, 442-449. https://doi.org/10.1002/ddr.21280 Arroyo-Lira, A.G., Rodríguez-Ramos, F., Chávez-Piña, A.E., 2014. Synergistic antinociceptive effect and gastric safety of the combination of docosahexaenoic acid and indomethacin in rats. Pharmacol. Biochem. Behav. 122, 74-81. https://doi.org/10.1016/j.pbb.2014.03.015 18

Arroyo-Lira, A.G., Rodríguez-Ramos, F., Ortiz, M.I., Castañeda-Hernández, G., Chávez-Piña, A.E., 2017. Supra-additive interaction of docosahexaenoic acid and naproxen and gastric safety on the formalin test in rats. Drug. Dev. Res. 78, 332-339. https://doi.org/10.1002/ddr.21396 Banafshe, H.R., Hamidi, G.A., Noureddini, M., Mirhashemi, S.M., Mokhtari, R., Shoferpour, M., 2014. Effect of curcumin on diabetic peripheral neuropathic pain: possible involvement of opioid system. Eur. J. Pharmacol. 723, 202206. https://doi.org/10.1016/j.ejphar.2013.11.033 Bang, S., Yoo, S., Yang. T.J., Cho, H., Hwang, S.W., 2012. 17(R)-resolvin D1 specifically inhibits transient receptor potential ion channel vanilloid 3 leading to peripheral antinociception. Br. J. Pharmacol. 165, 683-692. https://doi.org/10.1111/j.1476-5381.2011.01568.x Bang, S., Yoo, S., Yang, T.J., Cho, H., Kim, Y.G., Hwang, S.W., 2010. Resolvin D1 attenuates activation of sensory transient receptor potential channels leading to multiple anti-nociception. Br. J. Pharmacol. 161, 707-720. https://doi.org/10.1111/j.1476-5381.2010.00909.x Baron, R., Binder, A., Wasner, G., 2010. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol. 9, 807-819. https://doi.org/10.1016/S1474-4422(10)70143-5 Bento, A.F., Claudino, R.F., Dutra, R.C., Marcon, R., Calixto, J.B., 2011. Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirintriggered resolvin D1 and resolvin D2 prevent experimental colitis in mice. J. Immunol. 187, 1957-1969. https://doi.org/10.4049/jimmunol.1101305

19

Brigatte, P., Konno, K., Gutierrez, V.P., Sampaio, S.C., Zambelli, V.O., Picolo, G., Curi, R., Cury, Y. 2013. Peripheral kappa and delta opioid receptors are involved in the antinociceptive effect of crotalphine in a rat model of cancer pain. Pharmacol. Biochem. Behav. 109,1-7. https://doi.org/10.1016/j.pbb.2013.04.012 Carballo-Villalobos, A.I., González-Trujano, M.E., Pellicer, F., Alvarado-Vásquez, N., López-Muñoz, F.J., 2018. Central and peripheral anti-hyperalgesic effects of diosmin in a neuropathic pain model in rats. Biomed. Pharmacother. 97, 310-320. https://doi.org/10.1016/j.biopha.2017.10.077 Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods. 53, 55-63. PMID: 7990513 Chung, C., Carteret, A.F., McKelvy, A.D., Ringkamp, M., Yang, F., Hartke, T.V., Dong, X., Raja, S.N., Guan, Y. 2012. Analgesic properties of loperamide differ following systemic and local administration to rats after spinal nerve injury. Eur. J. Pain. 16, 1021-1032. https://doi.org/10.1002/j.15322149.2012.00148.x Dixon, W.J., 1980. Efficient analysis of experimental observations. Ann. Rev. Pharmacol. Toxicol. 20, 441-462. https://doi.org/10.1146/annurev.pa.20.040180.002301 Gamoh, S., Hashimoto, M., Sugioka, K., Shahdat Hossain, M., Hata, N., Misawa, Y., Masumura, S., 1999. Chronic administration of docosahexaenoic acid improves reference memory-related learning ability in young rats. Neuroscience. 93, 237-241. PMID: 10430487 20

Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 32, 77-88. PMID: 3340425 Heng, L.J., Qi, R., Yang, R.H., Xu, G.Z., 2015. Docosahexaenoic acid inhibits mechanical allodynia and thermal hyperalgesia in diabetic rats by decreasing the excitability of DRG neurons. Exp. Neurol. 271, 291-300. https://doi.org/10.1016/j.expneurol.2015.06.022 Huang, C.T., Tsai, Y.J., 2016. Docosahexaenoic acid confers analgesic effects after median nerve injury via inhibition of c-Jun N-terminal kinase activation in microglia. J. Nutritional Biochem. 29, 97-106. https://doi.org/10.1016/j.jnutbio.2015.11.009 Izquierdo, T., Espinoza de los Monteros-Zúñiga, A., Cervantes-Durán, C., Lozada, M.C., Godínez-Chaparro, B., 2013. Mechanisms underlying the antinociceptive effect of mangiferin in the formalin test. Eur. J. Pharmacol. 718, 393-400. https://doi.org/10.1016/j.ejphar.2013.08.004 Jensen, T.D., Baron, R., Haanpää, M., Kalso, E., Loeser, J.D., Rice, A.S., Treede, R.D., 2011. A new definition of neuropathic pain. Pain. 152, 2204-2205. https://doi.org/10.1016/j.pain.2011.06.017 Kapitzke, D., Vetter, I., Cabot, P.J., 2005. Endogenous opioid analgesia in peripheral tissues and the clinical implications for pain control. Ther. Clin. Risk Manag. 1, 279-297. PMCID: PMC1661636 Kishore, L., Kaur, N., Singh, R., 2018. Effect of Kaempferol isolated from seeds of Eruca sativa on changes of pain sensitivity in streptozotocin-induced

21

diabetic neuropathy. Inflammopharmacology. 26, 993-1003. http://doi.org/10.1007/s10787-017-0416-2 Ko, G.D., Nowacki, N.B., Arseneau, L., Eitel, M., Hum, A., 2010. Omega-3 fatty acids for neuropathic pain: case series. Clin. J. Pain. 26, 168-172. https://doi.org/10.1097/AJP.0b013e3181bb8533 Landa-Juárez, A.Y., Ortiz, M.I., Castañeda-Hernández, G., Chávez-Piña, A.E., 2016. Participation of potassium channels in the antinociceptive effect of docosahexaenoic acid in the rat formalin test. Eur. J. Pharmacol. 793, 95100. https://doi.org/10.1016/j.ejphar.2016.11.004 Liu, Z.H., Miao, G.S., Wang, J.N., Yang, C.X., Fu, Z.J., Sun, T., 2016. Resolvin D1 inhibits mechanical hypersensitivity in sciatica by modulating the expression of Nuclear Factor-κB, phospho-extracellular signal-regulated kinase, and pro- and antiinflammatory cytokines in the spinal cord and dorsal root ganglion. Anesthesiology. 124, 934-944. https://doi.org/10.1097/ALN.0000000000001010 Ma, J., Yu, H., Liu, J., Chen, Y., Wang, Q., Xiang, L., 2015. Metformin attenuates hyperalgesia an allodynia in rats with painful diabetic neuropathy induced by streptozotocin. Eur. J. Pharmacol. 764, 599-606. https://doi.org/10.1016/j.ejphar.2015.06.010 Manzhulo, I.V., Ogurtsova, O.S., Lamash, N.E., Latyshev, N.A., Kasyanov, S.P., Dyuizen, I.V., 2015. Analgetic effect of docosahexaenoic acid is mediated by modulating the microglia activity in the dorsal root ganglia in a rat model of neuropathic pain. Acta Histochem. 117, 659-666. https://doi.org/10.1016/j.acthis.2015.07.001 22

Mayurasakorn, K., Williams, J.J., Ten, V.S., Deckelbaum, R.J., 2011. Docosahexaenoic acid: brain accretion and roles in neuroprotection after brain hypoxia and ischemia. Curr. Opin. Clin. Nutr. Metab. Care. 14, 158167. https://doi.org/10.1097/MCO.0b013e328342cba5 Morales, M.E., Gereau, R.W., 2009. The effects of tail biopsy for genotyping on behavioral responses to nociceptive stimuli. PloS One. 4, e6457. https://doi.org/10.1371/journal.pone.0006457 Nakamoto, K., Nishinaka, T., Ambo, A., Mankura, M., Kasuya, F., Tokuyama, S., 2011. Possible involvement of β-endorphin in docosahexaenoic acidinduced antinociception. Eur. J. Pharmacol. 666, 100-104. https://doi.org/10.1016/j.ejphar.2011.05.047 Nakamoto, K., Nishinaka, T., Mankura, M., Fujita-Hamabe, W., Tokuyama, S., 2010. Antinociceptive effects of docosahexaenoic acid against various pain stimuli in mice. Biol. Pharm. Bull. 33, 1070-1072. PMID: 20522981 O´Brien, D.E., Alter, B.J., Satomoto, M., Morgan, C.D., Davidson, S., Vogt, S.K., Norman, M.E., Gereau, G.B., Demaro, J.A., Landreth, G.E., Golden, J.P., Gereau, R.W., 2015. ERK2 alone drives inflammatory pain but cooperates with ERK1 in sensory neuron survival. J. Neurosci. 35, 9491-9507. https://doi.org/10.1523/JNEUROSCI.4404-14.2015 Ocaña, M., Del Pozo, E., Baeyens, J.M., 1993. ATP-dependent K+ channel blockers antagonize morphine- but not U-504,88H-induced antinociception. Eur. J. Pharmacol. 230, 203-207. PMID: 8422902

23

Ortiz, M.I., Castañeda-Hernández, G., 2008. Examination of the interaction between peripheral lumiracoxib and opioids on the 1% formalin test in rats. Eur. J. Pain.12, 233–241. https://doi.org/10.1016/j.ejpain.2007.05.008 Ortiz, M.I., 2012. Metformin and phenformin block the peripheral antinociception induced by diclofenac and indomethacin on the formalin test. Life Sci. 90, 812. https://doi.org/10.1016/j.lfs.2011.10.009 Ortiz, M.I., Castro-Olguín, J., Peña-Samaniego, N., Castañeda-Hernández, G., 2005. Probable activation of the opioid receptor-nitric oxide-cyclic GMP-K+ channels pathway by codeine. Pharmacol. Biochem. Behav. 82, 695-703. https://doi.org/10.1016/j.pbb.2005.11.011 Ortiz, M.I., Fernández-Martínez, E., Ponce-Monter, H., Pérez-Hernández, N., Macías, A., Rangel-Flores, E., Castañeda-Hernández, G., 2007. The peripheral antinociceptive effect of nalbuphine is associated with activation of ATP-sensitive K+ channels. Proc. West. Pharm. Soc. 50, 72-74. PMID: 18605235 Ortiz, M.I., Ponce-Monter, H., Fernández-Martínez, E., Pérez-Hernández, N., Macías, A., Rangel-Flores, E., Castañeda-Hernández, G., 2007. Evaluation of the interaction between acemetacin and opioids on the hargreaves model of thermal hyperalgesia. Pharmacol. Biochem. Behav. 88, 47-54. https://doi.org/10.1016/j.pbb.2007.07.003 Pineda-Peña, E.A., Martínez-Pérez, Y., Galicia-Moreno, M., Navarrete, A., Segovia, J., Muriel, P., Favari, L., Castañeda-Hernández, G., Chávez-Piña, A.E., 2018. Participation of the anti-inflammatory and antioxidative activity of

24

docosahexaenoic acid on indomethacin-induced gastric injury model. Eur. J. Pharmacol. 818, 585-592. https://doi.org/10.1016/j.ejphar.2017.11.015 Quiñonez-Bastidas, G. N., Pineda-Farias, J.B., Flores-Murrieta, F.J., RodríguezSilverio, J., Reyes-García, J.G., Godínez-Chaparro, B., Granados-Soto, V., Rocha-González, H.I., 2018. Antinociceptive effect of (-)-epicatechin in inflammatory and neuropathic pain in rats. Behav. Pharmacol. 29, 270-279. https://doi.org/10.1097/FBP.0000000000000320 Redivo, D.D., Schreiber, A.K., Adami, E.R., Ribeiro, D.E., Joca, S.R., Zanoveli, J.M., Cunha, J.M., 2016. Effect of omega-3 polyunsaturated fatty acid treatment over mechanical allodynia and depressive-like behavior associated with experimental diabetes. Behav. Brain Res. 298, 57-64. https://doi.org/10.1016/j.bbr.2015.10.058 Rocha-González, H.I., Ramírez-Aguilar, M., Granados-Soto, V., Reyes-García, J.G., Torres-López, J.E., Huerta-Cruz, J.C., Navarrete, A., 2014. Antineuropathic effect of 7-hydroxy-3,4-dihydrocadalin in streptozotocininduced diabetic rodents. BMC. Complement. Altern. Med. 14,129. https://doi.org/10.1186/1472-6882-14-129 Salat, K., Gryzlo, B., Kulig, K., 2018. Experimental drugs for neuropathic pain. Curr. Neuropharmacol. 16, 1193-1209 https://doi.org/10.2174/1570159X16666180510151241 Sambasevam, Y., Omar Farouk, A.A., Tengku Mohamad, T.A., Sulaiman. M.R., Bharatham, B.H., Perimal, E.K., 2017. Cardamonin attenuates hyperalgesia and allodynia in a mouse model of chronic constriction injury-induced

25

neuropathic pain: possible involvement of the opioid system. Eur. J. Pharmacol. 796, 32-38. https://doi.org/10.1016/j.ejphar.2016.12.020 Silva, R.V., Oliveira, J.T., Santos, B.L.R., Dias, F.C., Martinez, A.M.B., Lima, C.K.F., Miranda, A.L.P., 2017. Long-chain Omega-3 fatty acids supplementation accelerates nerve regeneration and prevents neuropathic pain behavior in mice. Front. Pharmacol. 8, 723. https://doi.org/10.3389/fphar.2017.00723 Torres-Guzman, A.M., Morado-Urbina, C.E., Alvarado-Vazquez, P.A., AcostaGonzalez, R.I., Chávez-Piña, A.E., Montiel-Ruiz, R.M., Jimenez-Andrade, J.M., 2014. Chronic oral or intraarticular administration of docosahexaenoic acid reduces nociception and knee edema and improves functional outcomes in a mouse model of Complete Freund´s Adyuvant-induced knee arthritis. Arthritis Res. Ther. 16, R64. https://doi.org/10.1186/ar4502 Torres-López, J.E., Juárez-Rojop, I.E., Granados-Soto, V., Diaz-Zagoya, J.C., Flores-Murrieta, F.J., Ortíz-López, J.U., Cruz-Vera, J., 2007. Peripheral participation of cholecystokinin in the morphine-induced peripheral antinociceptive effect in non-diabetic and diabetic rats. Neuropharmacology. 52, 788-795. https://doi.org/10.1016/j.neuropharm.2006.09.015 Türkez, H., Geyikoglu, F., Yousef, M.I., 2012. Ameliorative effect of docosahexaenoic acid on 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced histological changes, oxidative stress, and DNA damage in rat liver. Toxicol. Ind. Health. 28, 687-696. https://doi.org/10.1177/0748233711420475

26

Wagner, K., Lee, K.S., Yang, J., Hammock, B.D., 2017. Epoxy fatty acids mediate analgesia in murine diabetic neuropathy. Eur. J. Pain. 21, 456-465. https://doi.org/10.1002/ejp.939 Woolf, C.J., 2004. Pain: moving from symptom control toward mechanism-specific pharmacologic management. Ann. Intern. Med. 140, 441-451. PMID: 15023710 Xu, Z.Z., Zhang, L., Liu, T., Park, J.T., Berta, T., Yang, R., Serhan, C.N., Ji, R.R., 2010. Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions. Nat. Med.16, 592-597. https://doi.org/10.1038/nm.2123 Yamamoto, H., Shimoshige, Y., Yamaji, T., Murai, N., Aoki, T., Matsuoka, N., 2009. Pharmacological characterization of standard analgesics on mechanical allodynia in streptozotocin-induced diabetic rats. Neuropharmacology. 57, 403-408. https://doi.org/10.1016/j.neuropharm.2009.06.037 Zhao, W.C., Zhang, B., Liao, M.J., Zhang, W.X., He, W.Y., Wang, H.B., Yang, C.X., 2014. Curcumin ameliorated diabetic neuropathy partially by inhibition of NADPH oxidase mediating oxidative stress in the spinal cord. Neurosci. Lett. 560, 81-85. https://doi.org/10.1016/j.neulet.2013.12.019 Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain.16, 109-10. PMID: 6877845

27

Fig. 1. The area under the curve (AUC) of the number of flinches during phase 1 and 2 on the formalin test in non-diabetic and diabetic rats. The bar plots show formalin-induced hyperalgesia during phase 1 (A) and phase 2 (B) in diabetic rats administered with streptozotocin (50 mg/kg, i.p.). Data are expressed as the mean ± S.E.M. of six to nine animals per group. *P < 0.05 from non-diabetic group determined by the Student’s t-test. Fig. 2. Dose-response curve of docosahexaenoic acid (DHA; 100–1000 μg/paw) and gabapentin (562–1778 μg/paw) in phase 2 of the formalin test in diabetic rats administered with streptozotocin (50 mg/kg, i.p.). Data are presented as mean ± S.E.M. of six to twelve animals per group. *P < 0.05 versus vehicle group by twoway ANOVA followed by Bonferroni’s post-hoc test. Fig. 3. Time course of the thermal hyperalgesia effect observed with 26% of active intensity in non-diabetic and diabetic rats. Each point corresponds to the mean ± S.E.M. of six animals per group. *P < 0.05 versus non-diabetic group determined by two-way ANOVA followed by Bonferroni’s post-hoc test. Fig. 4. The antihyperalgesic effect in the Hargreaves test of docosahexaenoic acid (DHA; 562 μg/paw) and gabapentin (1778 μg/paw). Data are expressed as the mean ± S.E.M. of six animals per group *P < 0.05 versus vehicle group. #P < 0.05 versus DHA group determined by one-way ANOVA followed by Newman-Keuls post-hoc test.

28

Fig. 5. Time course of paw withdrawal threshold obtained with non-diabetic, diabetic, docosahexaenoic acid (DHA; 562 μg/paw), and gabapentin (1778 μg/paw) diabetic rats’ groups. Values lower than 4 g indicate allodynia. Data are expressed as the mean ± S.E.M. of six animals per group *P < 0.05 versus diabetic rats determined by two-way ANOVA followed by Bonferroni’s post-hoc test. Fig. 6. Effect of local peripheral pre-treatment with the non-selective opioid receptor antagonist naloxone on docosahexaenoic acid (DHA; 562 μg/paw) induced-antinociception during phase 2 on the formalin test on diabetic rats. Data are expressed as the mean ± S.E.M. of six to nine animals per group *P < 0.05 versus vehicle, #P < 0.05 versus DHA treatment determined by one-way ANOVA followed by Newman-Keuls post-hoc test. Fig. 7. Effect of local peripheral pre-treatment with the selective δ receptor antagonist naltrindole on docosahexaenoic acid (DHA; 562 μg/paw) inducedantinociception during phase 2 on the formalin test on diabetic rats. Data are expressed as the mean ± S.E.M. of six animals per group *P < 0.05 versus vehicle, #

P < 0.05 versus DHA treatment determined by one-way ANOVA followed by

Newman–Keuls post-hoc test. Fig. 8. Effect of local peripheral pre-treatment with the selective μ receptor antagonist CTOP on docosahexaenoic acid (DHA; 562 μg/paw) inducedantinociception during phase 2 on the formalin test on diabetic rats. Data are expressed as the mean ± S.E.M. of six animals per group *P < 0.05 versus vehicle, #

P < 0.05 versus DHA treatment determined by one-way ANOVA followed by

Newman-Keuls post-hoc test. 29

Fig. 9. Effect of local peripheral pre-treatment with the selective κ receptor antagonist GNTI on docosahexaenoic acid (DHA; 562 μg/paw) inducedantinociception during phase 2 on the formalin test on diabetic rats. Data are expressed as the mean ± S.E.M. of six animals per group *P < 0.05 versus vehicle, #

P < 0.05 versus DHA treatment determined by one-way ANOVA followed by

Newman-Keuls post-hoc test.

30

Fig. 1.

31

Fig. 2.

32

Fig. 3.

Fig. 4. 33

Fig. 5.

34

Fig. 6.

35

Fig. 7.

36

Fig. 8.

37

Fig. 9.

38

Table 1. Body weight and blood glucose levels in rats after streptozotocin administration. Body weigth (g) Time

Blood glucose levels (mg/dl)

Non-diabetic

Diabetic

Non-diabetic

Diabetic

0

200 ± 2.20

200 ± 2.2

116.70 ± 3.31

119.2 ± 1.13

0.28

215 ± 3.20

212 ± 3.2

114.3 ± 3.73

392.1 ± 14.85 a

3

242.2 ± 4.25

234.1 ± 2.05

116.2 ± 1.99

474.6 ± 18.26 a

6

267.6 ± 5.73

250.9 ± 1.75 a

118.2 ± 2.57

478.3 ± 22.66 a

9

320.6 ± 6.92

251.8 ± 1.77 a

117.2 ± 1.62

547.5 ± 19.05 a

12

351.3 ± 4.64

253.1 ± 2.65 a

114.0 ± 2.69

526.0 ± 16.33 a

14

365.6 ± 3.15

255.1 ± 3.84 a

116.0 ± 2.90

550.8 ± 26.82 a

(weeks)

Data are expressed as mean ± S.E.M. of six to twelve animals. aP < 0.05 from nondiabetic group in the corresponding week by two-way ANOVA followed by Bonferroni´s pos-hoc test.

39