Elsevier

Experimental Neurology

Volume 279, May 2016, Pages 274-282
Experimental Neurology

Research Paper
CB2 cannabinoid receptor is involved in the anti-inflammatory effects of leptin in a model of traumatic brain injury

https://doi.org/10.1016/j.expneurol.2016.03.018Get rights and content

Highlights

  • TBI induces weight loss, neurological deficit and a potent neuroinflammatory response.

  • Leptin recovered parameters affected by TBI, including behavior and neuroinflammation.

  • CB2 receptor antagonist prevented some beneficial effects exerted by leptin after TBI.

Abstract

Background and purpose

The rates for traumatic brain injury (TBI) have risen in the last decade. Studies in animal models and clinical trials have not yet resulted in an effective treatment for TBI. Leptin, a 16 kDa peptidic hormone is mainly known as a regulator of energy balance and has been shown to exert neuroprotective effects in different models of brain pathology. In this study, we have assessed whether leptin exerts protective actions in a TBI mouse model. In addition, the possible implication of CB2 cannabinoid receptor in leptin actions has been explored, since it is known that the endocannabinoid system interacts with leptin and actively participates in brain recovery after lesions.

Methods

Swiss (CD1) male mice were subjected to weigh-drop model for TBI. Prior to the lesion, mice were injected with an antagonist of CB2 receptor (AM630) or the vehicle and immediately after TBI, they received leptin or vehicle treatment. Data were analyzed using a two-way ANOVA or the non-parametric test Kruskal–Wallis when appropriate. For correlation analyses, Spearman's rho test, followed by linear regression test, was used.

Results

TBI induced a neurological deficit, which was improved by leptin treatment. Leptin recovered several parameters affected by TBI, including the expression of cannabinoid receptors, axonal injury marker and neuroinflammatory components. The effects of leptin were prevented or reduced when it was administered in combination with the CB2 receptor antagonist, AM630.

Conclusions and implications

Since some of the beneficial effects of leptin were not evident in the presence of AM630, our results suggest that CB2 receptor might be involved in the full expression of the neuroprotective effects of the hormone. These findings open new avenues for the study of leptin as a therapeutic treatment for TBI and enhance the importance of CB2 receptor in TBI pathophysiology and recovery.

Graphical abstract

Schematic representation of molecular changes following TBI and the effect of leptin on brain trauma. Relative gene expression is represented below each condition with black rectangles. Naïve animals (A) present healthy neurons expressing high CB1 levels. Astrocytes and microglia stay at a resting state and express basal vimentin and CB2 levels respectively. Levels of IL-10 and plasma leptin are maintained constant. After TBI (B), neuron death occurs (black neuron) leading to decreased CB1 and axonal damage associated with increased BACE-1 expression. Glial cells change to a reactive phenotype characterized by high vimentin expression in astrocytes and increased CB2 levels in microglia, which release pro-inflammatory cytokines (TNF-alpha/TGF-beta) and reduce the production of anti-inflammatory molecules (IL-10). Plasma leptin also decreases. Leptin-treated mice (C) have less neuronal death which is reflected in recovered CB1 and BACE-1 expression. Astrocytes remain reactive and vimentin does not change. Leptin reduces microglia reactivity, which is related with a lower CB2 expression, TNF-alpha and TGF-beta and IL-10 levels are increased as also occurs with plasma leptin levels. (Images source: Mind the Graph platform).

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Introduction

According to the Centers for Diseases Control and Prevention, CDC (http://www.cdc.gov/traumaticbraininjury/data/rates), the rates for traumatic brain injury (TBI) have increased in the last ten years. It is important to note, that the incidence of TBI changes with age, increasing during adolescence (Bruns and Hauser, 2003). This peak could be related to an increase of risky behaviors in adolescents (Spear, 2000) which occurs in adolescent rodents that show elevated levels of novelty seeking (Adriani et al., 1998), impulsivity and risk-taking behavior (Adriani and Laviola, 2003). The causes of brain injuries also vary with age, and during adolescence, the leading causes of TBI are motor accidents and falls (Bruns and Hauser, 2003), which induce close-head injuries (85–89% of patients) (Masson et al., 2001, Wu et al., 2008). In order to mimic what happens in the clinical field, we studied close-head injuries using the weight-drop model (Cernak, 2005) in adolescent male mice. This is one of the most widely used methods to study TBI in rodents since it reproduces many of the symptoms found in TBI patients (Homsi et al., 2009).

Different therapeutic approaches, including anti-inflammatory molecules (Lopez-Rodriguez et al., 2015c, Schaible et al., 2013), hormones (Naderi et al., 2015, Tang et al., 2015), hyperbaric therapies (Chen et al., 2014, Wee et al., 2015) and repetitive transcranial magnetic stimulation have been tested for the treatment of TBI (Shin et al., 2014). In the last few years, several studies have shown that leptin exerts neuroprotective effects in different brain injury models both in vivo and in vitro (Dicou et al., 2001, Lu et al., 2006). Leptin is a 16 kDa peptidic hormone, which is primarily known as a regulator of food uptake and energy homeostasis (Coll et al., 2007, Friedman and Halaas, 1998). Since its discovery, many new functions, apart from controlling energy balance, have been ascribed to the hormone (Harvey, 2007, Tang, 2008). The reported beneficial effects of leptin in the central nervous system (CNS) include the enhancement of neuronal regeneration through JAK–STAT signaling (Tang, 2008), the attenuation of cerebral ischemia injury via the PI3K/Akt pathway (Zhang et al., 2013) and the reduction of infarct size in a murine model of photothrombosis of the distal middle cerebral artery (Avraham et al., 2010). These effects are mediated by the leptin receptor (ObR), a member of the class I cytokine receptor superfamily (Baumann et al., 1996). Its expression is not restricted to peripheral tissues, as it has also been detected in the CNS, including regions of the brain that are not directly related to energy homeostasis, such as the hippocampus, brainstem, cerebellum, cerebral cortex, amygdala and substantia nigra (Harvey, 2007). ObR is also expressed in T-cells, macrophages and neutrophils (Aguilar-Valles et al., 2015), which allows leptin to modulate immune responses by inhibiting apoptosis (Bruno et al., 2005), regulating the production of cytokines (Loffreda et al., 1998, Xiao et al., 2003) and improving survival during sepsis (Siegl et al., 2014, Tschöp et al., 2010).

Several studies have shown that the endocannabinoid system (ECS) participates in the resolution of brain injuries, decreasing vasoconstriction, gliosis, neuroinflammation and excitotoxicity (Arain et al., 2015, Shohami et al., 2011). Furthermore, the ECS plays an essential role during critical neurodevelopmental periods such as adolescence (Viveros et al., 2012). In previous studies using an adolescent TBI model, we have detected changes in the expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in the brain after injury and we have found that CB1 expression is negatively correlated with neurological deficit and edema, whereas CB2 expression is positively correlated with neurological impairment (Lopez-Rodriguez et al., 2015b). As Avraham et al. (2010) demonstrated in a phototrombosis model, there is a close relationship between leptin and the ECS, since the hormone modifies the expression of CB1 and CB2 after stroke. Additional evidence that relates leptin and the ECS is that CB2 receptor knock-out mice have reduced levels of circulating leptin (Deveaux et al., 2009). Moreover, the anorexic effect of leptin mediated by reactive oxygen species (ROS) is inhibited in the hypothalamus by a specific CB1 receptor agonist (Palomba et al., 2015). In addition, it is also known that the hormone is a negative regulator of the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) (Di Marzo et al., 2001).

Our group has recently demonstrated that CB1 and CB2 receptors are involved in the neuroprotective effects of estradiol (López Rodríguez et al., 2011) and minocycline (Lopez-Rodriguez et al., 2015c) in two different models of brain injury. Based on these previous findings, the aim of this study was to assess the potential beneficial effects of leptin in a murine model of TBI and to determine whether the ECS is involved in these effects. For this purpose, we used the weight-drop TBI model in adolescent male mice that were treated with leptin in the presence or absence of a CB2 receptor antagonist. We observed that leptin-treated mice recovered from the neurological deficit, and showed reduced neuroinflammatory molecules and increased anti-inflammatory cytokines. Some of these effects were not detected in the presence of the CB2 antagonist, which had an effect per se on β-secretase 1 (BACE-1) and tumor necrosis factor α (TNF-α) mRNA expression. The present results point to CB2 receptor as a mechanism underlying the response to TBI and open new avenues for the study of leptin as a potential treatment for these lesions.

Section snippets

Animals

All the experiments were performed in adolescent (post-natal day (pnd) 35–36) Swiss (CD1) male mice (Harlan, Spain). Animals were housed in a controlled temperature environment (22 ± 2 °C), 12 h light/dark cycle and with access to food and water ad libitum. Animal care and procedures were approved by our institutional animal use and care committee (Comité de Experimentación Animal CEA-UCM; 68/2012) and followed the European Communities Council Directive (2010/63/EU) and the Spanish regulations (Ley

Results

Noninjured animals (naïve) were included in this study to verify that the TBI model had an effect and to normalize the mRNA levels in the qRT-PCR analysis. Student's t-test comparisons between naïve and TBI-vehicle animals showed that after lesion, there was a decrease in body weight [t = 0.000] which was accompanied by a decrease in neurological score, indicative of neurological deficit [t = 0.000] (Fig. 1).

We also measured other biochemical parameters that are known to be affected by TBI,

Discussion

In this study, we have observed that leptin reduces some of the parameters altered by TBI in adolescent mice by a mechanism that partially involves CB2 cannabinoid receptors. For this study we used a close-head model of TBI since the majority of the cases of TBI in humans correspond to close-head injuries (Masson et al., 2001, Wu et al., 2008). We used adolescent mice since there is a high incidence of TBI in adolescent humans due to an increase in the need for novelty seeking or risk taking

Conclusions

In summary, we found that leptin treatment induced changes after TBI in several parameters including neurological deficit, cannabinoid receptors, axonal injury marker and neuroinflammatory components. In several cases, there was a significant interaction between leptin and AM630, suggesting a pharmacological interaction, i.e., in these cases CB2 receptor activity might be necessary for the full expression of the protective mechanisms elicited by the hormone. The present findings open new

Competing interest

None of the authors have any competing interests in the manuscript.

Acknowledgments

We acknowledge financial support from the Ministerio de Economía y Competitividad, Spain (BFU2011-30217-C03-01 and BFU2012-38144), Redes temáticas de Investigación Cooperativa en Salud, Red de Trastornos Adictivos (RD2012/0028/0021-FEDER), GRUPO UCM (951579) and Precipita-FECYT Crowd funding program.

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