Pathophysiological roles of thrombospondin-4 in disease development.

Thrombospondin-4 (TSP-4) belongs to the extracellular matrix glycoprotein family of thrombospondins (TSPs). The multidomain, pentameric structure of TSP-4 allows its interactions with numerous extracellular matrix components, proteins and signaling molecules that enable its modulation to various physiological and pathological processes. Characterization of TSP-4 expression under development and pathogenesis of disorders has yielded important insights into mechanisms underlying the unique role of TSP-4 in mediating various processes including cell-cell, cell-extracellular matrix interactions, cell migration, proliferation, tissue remodeling, angiogenesis, and synaptogenesis. Maladaptation of these processes in response to pathological insults and stress can accelerate the development of disorders including skeletal dysplasia, osteoporosis, degenerative joint disease, cardiovascular diseases, tumor progression/metastasis and neurological disorders. Overall, the diverse functions of TSP-4 suggest that it may be a potential marker or therapeutic target for prognosis, diagnosis, and treatment of various pathological conditions upon further investigations. This review article highlights recent findings on the role of TSP-4 in both physiological and pathological conditions with a focus on what sets it apart from other TSPs.

The role of the anterior pretectal nucleus in pain modulation: A comprehensive review.

Descending pain modulation involves multiple encephalic sites and pathways that range from the cerebral cortex to the spinal cord. Behavioral studies conducted in the 1980s revealed that electrical stimulation of the pretectal area causes antinociception dissociation from aversive responses. Anatomical and physiological studies identified the anterior pretectal nucleus and its descending projections to several midbrain, pontine, and medullary structures. The anterior pretectal nucleus is morphologically divided into a dorsal part that contains a dense neuron population (pars compacta) and a ventral part that contains a dense fiber band network (pars reticulata). Connections of the two anterior pretectal nucleus parts are broad and include prominent projections to and from major encephalic systems associated with somatosensory processes. Since the first observation that acute or chronic noxious stimuli activate the anterior pretectal nucleus, it has been established that numerous mediators participate in this response through distinct pathways. Recent studies have confirmed that at least two pain inhibitory pathways are activated from the anterior pretectal nucleus. This review focuses on rodent anatomical, behavioral, molecular, and neurochemical data that have helped to identify mediators of the anterior pretectal nucleus and pathways related to its role in pain modulation.

Cannabidiol induces antidepressant and anxiolytic-like effects in experimental type-1 diabetic animals by multiple sites of action.

Cannabidiol (CBD), a phytocannabinoid compound, presents antidepressant and anxiolytic-like effects in the type-1 diabetes mellitus(DM1) animal model. Although the underlying mechanism remains unknown, the type-1A serotonin receptor (5-HT1A) and cannabinoids type-1 (CB1) and type-2 (CB2) receptors seem to play a central role in mediating the beneficial effects on emotional responses. We aimed to study the involvement of these receptors on an antidepressant- and anxiolytic-like effects of CBD and on some parameters of the diabetic condition itself. After 2 weeks of the DM1 induction in male Wistar rats by streptozotocin (60 mg/kg; i.p.), animals were treated continuously for 2-weeks with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg, i.p.), CB1 antagonist AM251 (1 mg/kg i.p.) or CB2 antagonist AM630 (1 mg/kg i.p.) before the injection of CBD (30 mg/kg, i.p.) or vehicle (VEH, i.p.) and then, they were submitted to the elevated plus-maze and forced swimming tests. Our findings show the continuous treatment with CBD improved all parameters evaluated in these diabetic animals. The previous treatment with the antagonists - 5-HT1A, CB1, or CB2 - blocked the CBD-induced antidepressant-like effect whereas only the blockade of 5-HT1A or CB1 receptors was able to inhibit the CBD-induced anxiolytic-like effect. Regarding glycemic control, only the blockade of CB2 was able to inhibit the beneficial effect of CBD in reducing the glycemia of diabetic animals. These findings indicated a therapeutic potential for CBD in the treatment of depression/anxiety associated with diabetes pointing out a complex intrinsic mechanism in which 5-HT1A, CB1, and/or CB2 receptors are differently recruited.

Antinociceptive action of cannabidiol on thermal sensitivity and post-operative pain in male and female rats.

This study investigated the antinociceptive potential of cannabidiol (CBD) in male and female Wistar rats. The assessment and analysis included tail withdrawal to thermal stimulation (tail flick test) and mechanical allodynia induced by plantar incision injury (von Frey test). CBD reduced acute thermal sensitivity in uninjured animals and post-operative mechanical allodynia in males and females. In the tail flick test, CBD 30 mg/kg i.p. was required to induce antinociception in males. During the proestrus phase, females did not show a statistically significant antinociceptive response to CBD treatment despite a noticeable trend. In contrast, in a separate group of rats tested during the late diestrus phase, antinociception varied with CBD dosage and time. In the post-operative pain model, CBD at 3 mg/kg decreased mechanical allodynia in males. Similarly, this dose reduced allodynia in females during proestrus. However, in females during late diestrus, the lower dose of CBD (0.3 mg/kg) reduced mechanical allodynia, although the latency to onset of the effect was slower (90 min). The effectiveness of a 10-fold lower dose of CBD during the late diestrus stage in females suggests that ovarian hormones can influence the action of CBD. While CBD has potential for alleviating pain in humans, personalized dosing regimens may need to be developed to treat pain in women.

Pharmacological Interaction Between Cannabidiol and Tramadol on Experimental Diabetic Neuropathic Pain: An Isobolographic Analysis.

Introduction: Diabetic neuropathies are the most prevalent chronic complications of diabetes, characterized by pain and substantial morbidity. Although many drugs have been approved for the treatment of this type of pain, including gabapentin, tramadol (TMD), and classical opioids, it is common to report short-term results or potentially severe side effects. TMD, recommended as a second-line treatment can lead to unwanted side effects. Cannabidiol (CBD) has been gaining attention recently due to its therapeutic properties, including pain management. This study aimed to characterize the pharmacological interaction between CBD and TMD over the mechanical allodynia associated with experimental diabetes using isobolographic analysis. Materials and Methods: After diabetes induction by streptozotocin (STZ), diabetic rats were systemically treated with CBD or TMD alone or in combination (doses calculated based on linear regression of effective dose 40% [ED40]) and had the mechanical threshold evaluated using the electronic Von Frey apparatus. Both experimental and theoretical additive ED40 values (Zmix and Zadd, respectively) were determined for the combination of CBD plus TMD in this model. Results: Acute treatment with CBD (3 or 10 mg/kg) or TMD (2.5, 5, 10, or 20 mg/kg) alone or in combination (0.38+1.65 or 1.14+4.95 mg/kg) significantly improved the mechanical allodynia in STZ-diabetic rats. Isobolographic analysis revealed that experimental ED40 of the combination (Zmix) was 1.9 mg/kg (95% confidence interval [CI]=1.2-2.9) and did not differ from the theoretical additive ED40 2.0 mg/kg (95% CI=1.5-2.8; Zadd), suggesting an additive antinociceptive effect in this model. Conclusions: Using an isobolographic analysis, these results provide evidence of additive pharmacological interaction between CBD and TMD over the neuropathic pain associated with experimental diabetes induced by STZ.

Cannabidiol enhances the antinociceptive effects of morphine and attenuates opioid-induced tolerance in the chronic constriction injury model.

Neuropathic pain (NP) is a complex health problem that includes sensorial manifestations such as evoked and ongoing pain. Cannabidiol (CBD) has shown potential in the treatment of NP and the combination between opioids and cannabinoids has provided promising results on pain relief. Thus, our study aimed to investigate the effect of treatment combination between CBD and morphine on evoked and ongoing pain, and the effect of CBD on morphine-induced tolerance in the model of chronic constriction injury (CCI) of the sciatic nerve in rats. Mechanical thresholds (i.e., evoked pain) were evaluated before and 7 days after surgery. We also employed a 4-day conditioned place preference (CPP) protocol, to evaluate relief of ongoing pain (6-9 days after surgery). Treatment with morphine (2 and 4 mg/kg) or CBD (30 mg/kg) induced a significant antinociceptive effect on evoked pain. The combination of CBD (30 mg/kg) and morphine (1 mg/kg) produced an enhanced antinociceptive effect, when compared to morphine alone (1 mg/Kg). Treatment with morphine (1 and 2 mg/kg) or CBD (30 mg/kg) alone failed to induce significant scores in the CPP test. However, combined treatment of CBD (30 mg/kg) and morphine (1 mg/kg) provided significant positive scores, increased the number of entrances in the drug-paired chamber in the CPP test and did not alter locomotor activity in rats. Lastly, treatment with CBD partially attenuated morphine-induced tolerance. In summary, our results support the indication of CBD as an adjuvant to opioid therapy for the attenuation of NP and opioid-induced analgesic tolerance.

Sex-dependent differences in the anxiolytic-like effect of cannabidiol in the elevated plus-maze.

RATIONALE: Cannabidiol (CBD), the major non-psychoactive constituent of cannabis, has therapeutic potential for the treatment of anxiety. Most preclinical studies investigate only acute effects of CBD and only in males, yet the drug is most likely to be used over a sustained period in clinical practice. OBJECTIVES: The objectives of this study were to investigate the anxiolytic-like effect of CBD in female rats compared to males and to determine whether the responsiveness of females was influenced by the stage of the estrous cycle. METHODS: We carried out experiments to compare the effect of CBD in male and female rats in the elevated plus maze (EPM) in response to acute and short-term (4 days) administration through a complete cycle in females. RESULTS: Male and female rats behaved in a similar manner in the EPM, but females in the late diestrus (LD) phase exhibited more anxiety-like behavior than at other stages, the difference reaching statistical significance compared to proestrus stages. CBD produced anxiolytic-like effects in both sexes, but female rats were responsive only in LD and 10-fold lower dose than males. After sub-chronic (4 days) treatment, responsiveness to CBD was maintained in females in LD, but females in proestrus remained unresponsive to CBD treatment. CONCLUSIONS: We suggest that there are sex differences in the anxiolytic-like effects of CBD in rats that reflect different underlying mechanisms: based on literature data, gonadal hormone status linked to GABAA receptor expression in females, and 5-HT1A receptor activation in males.

Influence of estrous cycle stage on acquisition and expression of fear conditioning in female rats.

It is unclear whether all animal models of anxiety-like states developed using males are appropriate for use in females. In females, tests involving a learning component might be influenced not only by estrous cycle stage on the test day but also by the stage during the conditioning process. We used two tests - conditioned freezing (CF) and fear potentiated startle (FPS) to compare responsiveness of male rats and females conditioned and/or tested in proestrus (P) or late diestrus (LD). For CF all rats displayed a similar freezing response regardless of sex or estrous cycle stage. In terms of FPS, males and females conditioned in P and tested in P or LD, and females conditioned in LD and tested in LD all showed potentiated startle. The response waned during the test session in males and in females conditioned in P, but not in those conditioned in LD. In contrast, FPS was not apparent in the first half of the test session in females conditioned in LD and tested in P but developed in the second half. We suggest that fear learning during P and LD is robust but may be initially be obscured in rats tested in P because of generalization to the CS due to high estrogen. Estrous cycle stage is an important consideration which must be taken into account in designing behavioural tests in females.

Two-weeks treatment with cannabidiol improves biophysical and behavioral deficits associated with experimental type-1 diabetes.

The prevalence rates of depression and anxiety are at least two times higher in diabetic patients, increasing morbidity and mortality. Cannabidiol (CBD) has been identified as a therapeutic agent viable to treat diverse psychiatric disorders. Thus, this study aimed to investigate the effect of CBD treatment (once a day for 14 days starting two weeks after diabetes induction; at doses of 0, 3, 10 or 30 mg/kg, i.p.) on depression- and anxiety-like behaviors associated with experimental diabetes induced by streptozotocin (60 mg/kg; i.p.) in rats. Levels of plasma insulin, blood glucose, and weight gain were evaluated in all experimental groups, including a positive control group treated with imipramine. The rats were tested in the modified forced swimming test (mFST) and elevated plus maze (EPM) test. Besides, the levels of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) in two emotion-related brain regions, the prefrontal cortex (PFC) and hippocampus (HIP) were evaluated using high-pressure liquid chromatography. Our results showed that CBD treatment (only at the higher dose of 30 mg/kg) reduced the exaggerated depressive- and anxiogenic-like behaviors of diabetic (DBT) rats, which may be associated with altered 5-HT, NA and/or DA levels observed in the PFC and HIP. Treatment with CBD (higher dose) also induced a significant increase in weight gain and the insulin levels (and consequently reduced glycemia) in DBT rats. The long-term CBD effects gave rise to novel therapeutic strategies to limit the physiological and neurobehavioral deficits in DBT rats. This approach provided evidence that CBD can be useful for treating psychiatry comorbidities in diabetic patients.

The antinociceptive effect of anterior pretectal nucleus stimulation is mediated by distinct neurotransmitter mechanisms in descending pain pathways.

Electrical stimulation of the anterior pretectal nucleus (APtN) activates two descending pain inhibitory pathways. One of these pathways relays in the ipsilateral lateral paragigantocellular nucleus (LPGi), whereas the other pathway relays in the contralateral pedunculopontine tegmental nucleus (PPTg). Antinociceptive effect of APtN stimulation has been seen in various pain models in the rodents. Similarly, LPGi or PPTg stimulation results in higher pain thresholds. Descending antinociceptive pathways activated by electrical APtN stimulation have been elucidated, but the underlying neurotransmitter mechanisms involved have not been clarified yet. This study investigates the role that endogenous signaling plays in the ipsilateral LPGi or contralateral PPTg after the APtN is stimulated in the tail-flick test. First, we submitted rats to excitotoxic injection of N-methyl-d-aspartate (NMDA) into the contralateral PPTg. Then, we examined whether blockage of NMDA (AP-7), serotonergic (methysergide), or opioid (naloxone) receptors in the ipsilateral LPGi is required for APtN stimulation-evoked analgesia (SEA). Likewise, we examined the effects of antagonists of NMDA, serotonergic, or cholinergic nicotinic (mecamylamine) receptors on the contralateral PPTg in ipsilateral LPGi-lesioned rats. Our results confirmed that APtN stimulation activates two pain inhibitory pathways and showed that endogenous opioid signaling in the ipsilateral LPGi appears to be necessary for APtN SEA and for endogenous NMDA, serotoninergic, and nicotinergic signaling in the contralateral PPTg.

Cannabidiol attenuates mechanical allodynia in streptozotocin-induced diabetic rats via serotonergic system activation through 5-HT1A receptors.

Most diabetic patients describe moderate to severe pain symptoms whose pharmacological treatment is palliative and poorly effective. Cannabidiol (CBD) has shown promising results in painful conditions. Then, we aimed to investigate the potential antinociceptive effect of CBD over the mechanical allodynia in streptozotocin-induced diabetic (DBT) rats, as well as its involved mechanisms. Wistar adult male diabetic rats were treated acutely or sub-chronically (for 14 days) with CBD (0.1, 0.3 or 3 mg/kg, intraperitoneal; i.p.) and had their mechanical threshold assessed using the electronic Von Frey. Acute treatment with CBD (at doses of 0.3 and 3 mg/kg) exerted a significant anti-allodynic effect, which is not associated with locomotor impairment. The antinociceptive effect of CBD (3 mg/kg) was not altered by the pre-treatment with CB1 or CB2 receptor antagonists (AM251 and AM630; respectively; both at a dose of 1 mg/kg, i.p.) nor by glycine receptor antagonist (strychnine hydrochloride, 10 µg/rat, intrathecal, i.t.). However, this effect was completely prevented by the pre-treatment with the selective 5-HT1A receptor antagonist WAY 100135 (3 µg/rat, i.t.). Sub-chronic treatment with CBD (0.3 or 3 mg/kg) induced a sustained attenuation of the mechanical allodynia in DBT rats. DBT rats presented significantly lower spinal cord levels of serotonin, which was prevented by the daily treatment with CBD (0.3 mg/kg). Taken together, our data suggest that CBD may be effective in the treatment of painful diabetic neuropathy and this effect seems to be potentially mediated by the serotonergic system activation through 5-HT1A receptors.

Sub-chronic treatment with cannabidiol but not with URB597 induced a mild antidepressant-like effect in diabetic rats.

Depression associated with diabetes has been described as a highly debilitating comorbidity. Due to its complex and multifactorial mechanisms, the treatment of depression associated with diabetes represents a clinical challenge. Cannabidiol (CBD), the non-psychotomimetic compound derived from Cannabis sativa, has been pointed out as a promising compound for the treatment of several psychiatric disorders. Here, we evaluated the potential antidepressant-like effect of acute or sub-chronic treatment with CBD in diabetic rats using the modified forced swimming test (mFST). Also, to better understand the functionality of the endocannabinoid system in diabetic animals we also evaluated the effect of URB597, a fatty acid amide hydrolase inhibitor. Four weeks after the treatment with streptozotocin (60 mg/kg; i.p.; diabetic group-DBT) or citrate buffer (i.p.; normoglycemic group-NGL), DBT animals received an acute intraperitoneal injection of CBD (0, 0.3, 3, 10, 30 or 60 mg/kg), 1 h before the mFST, or URB597 (0, 0.1, 0.3 or 1 mg/kg) 2 h before the mFST. In another set of experiments, animals were sub-chronically treated with CBD (0, 0.3, 3, 30 or 60 mg/kg i.p.), 24, 5 and 1 h before the mFST or URB597 (0, 0.1, 0.3 or 1 mg/kg i.p.) 24, 5 and 2 h before the mFST. The NGL group was acutely treated with CBD (0, 30 mg/kg i.p.) or URB597 (0, 0.3 mg/kg; i.p.). Acute treatment with either CBD or URB induced an antidepressant-like effect in NGL rats, but not in DBT rats. However, sub-chronic treatment with CBD (only at a dose of 30 mg/kg), but not with URB597, induced a mild antidepressant-like effect in DBT animals. Neither body weight nor blood glucose levels were altered by treatments. Considering the importance of the endocannabinoid system to the mechanism of action of many antidepressant drugs, the mild antidepressant-like effect of the sub-chronic treatment with CBD, but not with URB597 does not invalidate the importance of deepening the studies involving the endocannabinoid system particularly in DBT animals.

Angiotensin AT1 receptors modulate the anxiogenic effects of angiotensin (5-8) injected into the rat ventrolateral periaqueductal gray.

Losartan and PD 123,319 are non-peptide angiotensin (Ang) receptor antagonists for the AT1 and AT2 subtypes of Ang II receptors, respectively. The tetrapeptide Ang (5-8) is the smallest Ang-peptide that elicits anxiogenic effects on unconditioned and conditioned experimental models upon injection into the ventrolateral column of the periaqueductal gray (vlPAG), and Ang (5-8) can be synthesized (from Ang II or Ang III) and inactivated in this mesencephalic structure. The vlPAG is also known to play a central role in mechanisms of fear and anxiety. We therefore utilized male Wistar rats to examine the effects of losartan and PD 123,319 injections, selective antagonists of the AT1 and AT2 receptors, respectively, into the vlPAG in the elevated plus-maze, a classic rat model of anxiety, and against the anxiogenic effect of Ang (5-8) (0.4 nmol/0.25µL) upon injection into the same region. The anxiolytic profile was dependent on the dose of intra-vlPAG losartan, whereas no effects on experimental anxiety were observed in the plus-maze following PD 123,319 injection. The anxiogenic effect of Ang (5-8) injection into the vlPAG remained unchanged in the PD 123,319-pretreated rats, but the effect did not occur in losartan-pretreated rats. The results led us to suggest that the anxiogenic effect of Ang (5-8) injection into the vlPAG may depend on the local activation of AT1, but not AT2 receptors. Activation of AT1 receptors in structures nearby vlPAG may be tonically involved in fear and experimental anxiety.

Cannabidiol Is a Potential Therapeutic for the Affective-Motivational Dimension of Incision Pain in Rats.

Background: Pain involves different brain regions and is critically determined by emotional processing. Among other areas, the rostral anterior cingulate cortex (rACC) is implicated in the processing of affective pain. Drugs that interfere with the endocannabinoid system are alternatives for the management of clinical pain. Cannabidiol (CBD), a phytocannabinoid found in Cannabis sativa, has been utilized in preclinical and clinical studies for the treatment of pain. Herein, we evaluate the effects of CBD, injected either systemically or locally into the rACC, on mechanical allodynia in a postoperative pain model and on the negative reinforcement produced by relief of spontaneous incision pain. Additionally, we explored whether CBD underlies the reward of pain relief after systemic or rACC injection. Methods and Results: Male Wistar rats were submitted to a model of incision pain. All rats had mechanical allodynia, which was less intense after intraperitoneal CBD (3 and 10 mg/kg). Conditioned place preference (CPP) paradigm was used to assess negative reinforcement. Intraperitoneal CBD (1 and 3 mg/kg) inverted the CPP produced by peripheral nerve block even at doses that do not change mechanical allodynia. CBD (10 to 40 nmol/0.25 µL) injected into the rACC reduced mechanical allodynia in a dose-dependent manner. CBD (5 nmol/0.25 µL) did not change mechanical allodynia, but reduced peripheral nerve block-induced CPP, and the higher doses inverted the CPP. Additionally, CBD injected systemically or into the rACC at doses that did not change the incision pain evoked by mechanical stimulation significantly produced CPP by itself. Therefore, a non-rewarding dose of CBD in sham-incised rats becomes rewarding in incised rats, presumably because of pain relief or reduction of pain aversiveness. Conclusion: The study provides evidence that CBD influences different dimensions of the response of rats to a surgical incision, and the results establish the rACC as a brain area from which CBD evokes antinociceptive effects in a manner similar to the systemic administration of CBD. In addition, the study gives further support to the notion that the sensorial and affective dimensions of pain may be differentially modulated by CBD.

Neural Correlates of the Antinociceptive Effects of Stimulating the Anterior Pretectal Nucleus in Rats.

Stimulation-evoked antinociception (SEA) from the anterior pretectal nucleus (APtN) activates mechanisms that descend to the spinal cord through the dorsolateral funiculus, but the encephalic route followed by the descending pathways from the APtN is not completely known. This study evaluated the changes in the SEA from the APtN in the Wistar rat tail-flick test after lidocaine-induced neural block or N-methyl-d-aspartate-induced neurotoxic lesion of the deep mesencephalic nucleus (DpMe), tegmental pedunculopontine nucleus (PPTg), or lateral paragigantocellular nucleus (LPGi). The SEA from the APtN was less intense after neural block of the contralateral DpMe or PPTg or the ipsilateral LPGi, but was not changed by the neural block of the ipsilateral DpMe or PPTg or the contralateral LPGi. Antinociception did not occur when APtN stimulation was carried out 5 minutes after lidocaine or 6 days after N-methyl-d-aspartate injections into the contralateral DpMe and the ipsilateral LPGi, or into the contralateral PPTg and the ipsilateral LPGi. We conclude that the SEA from the APtN activates 2 descending pain inhibitory pathways, one relaying in the ipsilateral LPGi and another relaying sequentially in the contralateral DpMe and PPTg. PERSPECTIVE: The antinociceptive effect of the APtN stimulation involves 2 descending pathways: one relaying in the ipsilateral LPGi and another descending contralaterally via relays in the DpMe and PPTg.

Effects of angiotensin (5-8) microinfusions into the ventrolateral periaqueductal gray on defensive behaviors in rats.

Peptides of the renin-angiotensin system modulate blood pressure and hydro-electrolyte composition. Angiotensin (Ang) receptors are localized in brain areas related to the regulation of autonomic and endocrine control and involved in sensory perception, memory process and behavioral responses. Among these areas, the ventrolateral periaqueductal gray (vlPAG) is one of the most important structures of the neuronal circuitry controlling the autonomic and behavioral components of emotional states. Although Ang II metabolism in the vlPAG forms several Ang-peptides including Ang (5-8), the role of this tetrapeptide in the organization of defensive responses has not yet been described. To address this issue, the purpose of the present study was to determine the effects of intra-vlPAG injections of Ang (5-8) (0.2, 0.4 and 0.8 nmol/0.25 µL) in rats submitted to the elevated plus-maze (EPM) test. Additionally, it was evaluated the effects of intra-vlPAG Ang (5-8) on the expression of conditioned fear, assessed by the fear-potentiated startle and contextual conditioned freezing tests. The results showed that Ang (5-8) produced an intense, dose-related reduction in the entries into and time spent in the open arms of the EPM, decreased direct exploration and increased risk assessment behaviors. Moreover, intra-vlPAG injections of Ang (5-8) before the test session promoted pro-aversive effects in the FPS and enhanced contextual freezing. Taken together, these results point out to an important anxiogenic-like action for Ang (5-8) in the mediation of defensive behaviors organized in the vlPAG.