Page 1 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1 Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury
*
*
Marika Cordaro 1, Daniela Impellizzeri 1, Irene Paterniti1, Giuseppe Bruschetta1, Rosalba Siracusa1, Daniela De Stefano2, Salvatore Cuzzocrea1,3, Emanuela Esposito1
1.
Department of Biological and Environmental Sciences, University of Messina, Messina
2.
Department of Experimental Pharmacology University of Naples Federico II, Naples
3.
Manchester Biomedical Research Centre, Manchester Royal Infirmary, School of Medicine, University of Manchester, UK
*the authors contributed equally to this work
Cuzzocrea Salvatore, Full Professor.email: [email protected] Esposito Emanuela, Researcher Assistant. email: [email protected] De Stefano Daniela, Researcher Assistant. email: [email protected] Paterniti Irene, Post Doc. email: [email protected] Impellizzeri Daniela, Post Doc. email: [email protected] Cordaro Marika, PhD Student. email: [email protected] Bruschetta Giuseppe, PhD Student. email: [email protected] Siracusa Rosalba, Resident Student. email [email protected]
Corresponding Author: Prof. Salvatore Cuzzocrea - Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres, 31 – 98166 Messina, Italy; Phone: +390906765208; email: [email protected]
Running Title: Co-ultraPEALut and traumatic brain injury
Page 2 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Abstract Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. In the present study, we performed a widely-used model of TBI to determine the neuroprotective propriety of palmitoylethanolamide (PEA) and the antioxidant effect of a flavonoid luteolin (Lut), given as a co-ultramicronized compound Co-ultraPEALut. We demonstrated that the treatment with Co-ultraPEALut resulted in a significant improvement of motor and cognitive recovery after controlled cortical impact (CCI), as well as markedly reducing lesion volumes. Moreover, our results revealed the ability of Co-ultraPEALut, to reduce brain trauma through modulation of NF-κB activation. In addition, treatment with Co-ultraPEALut significantly enhanced the post-TBI expression of the neuroprotective neurotrophins GDNF compared to vehicle. Co-ultraPEALut at the dose of 1 mg/kg, also modulated apoptosis, the release of cytokine and ROS, the activation of chymase, tryptase and nitrotyrosine and inhibited autophagy. Thus, our data demonstrated that Co-ultraPEALut at a lower dose compared to PEA alone, can exert neuroprotective effects and the combination of both could improve their ability to counteract the neurodegeneration and neuroinflammation induced by TBI.
Keywords Neuroprotection, palmitoylethanolamide, luteolin, inflammation, autophagy
Page 3 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 Introduction Traumatic brain injury (TBI) is a major public health problem and a leading cause of mortality and morbidity worldwide, particularly among the young.1,
2
The American Brain Injury Association
describes TBI as “an alteration in brain function, or other evidence of brain pathology. caused by an external force”.3 TBI is a highly complex disorder that includes varying degrees of contusion, diffuse axonal injury, haemorrhage and hypoxia, defined primary injury.1, 4 Collectively, these effects induce biochemical and metabolic changes that lead to progressive tissue damage and associated cell death, called second injury.5 TBI induces a strong inflammatory response characterized by the recruitment of peripheral leukocytes and enhanced chymase, tryptase as well as nitrotyrosine activation into the cerebral parenchyma.6-8 Moreover, activated microglia migrates to injured sites and releases cytokines, chemotactic cytokines, reactive oxygen species (ROS), nitric oxide, proteases and other factors with cytotoxic effects, which may in turn exacerbate neuronal death.9, 10 Also, a close relationship exists between the degree of oxidative stress and the pathogenesis of TBI.11 Enhanced production of ROS and reactive nitrogen species (RNS) causes oxidative/nitrosative stress
leading to damage in
lipids,12, 13 proteins and nucleic acids.14, 15 Additionally, Lai et colleagues, have demonstrated that oxidative stress contributes to overall autophagy in mice after TBI and that this pathway was partially inhibited by an antioxidant.16 Autophagy, from Greek “self-eating” is a major protein degradation system that targets primarily long-lived cytoplasmic proteins.17 Autophagy involves a dynamic subcellular membrane rearrangement whereby cytoplasmic proteins and organelles are sequestered in double-membrane vesicular structures (autophagosomes) that fuse with the lysosome or vacuole, where the material is catabolically degraded, recycled and utilized as an energy source.18 Several key molecular components participate in the initiation, progression, and completion of autophagy, like the mammalian target of rapamycin (mTOR) that inhibits autophagy whereas Beclin 1 and LC3 promote it.
Page 4 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 In recent years, the pathophysiology of TBI has been a focus of extensive studies; animal models have been proved to be important tools in this field, and are employed to investigate the mechanisms of primary and secondary injury. Evaluation of improvement in neurological function using a clinically relevant TBI model such as controlled cortical impact (CCI) establishes efficacy and clinical relevance of an experimental therapy.8 In the CCI model, the injury to the brain initially presents as necrotic cell death in the underlying tissue and white matter axonal injury, both reminiscent of the clinical TBI pathology, but also followed by apoptotic cell death in surrounding tissue due to multiple subsequent events such as edema, ischemia, excitotoxicity and altered gene expression.19 Our previous works clearly demonstrated that treatment with palmitoylethanolamide (PEA) at 10 mg/kg significantly reduced the inflammation process associated with experimental spinal cord injury (SCI) 20 and TBI mice models.8 PEA is abundant in the central nervous system (CNS), and it is conspicuously produced by glial cells.21-23 It has been shown to inhibit peripheral inflammation and mast cell degranulation
24
, as
well as exert antinociceptive effects in rats and mice.25, 26 However, PEA lacks a direct antioxidant capacity to prevent the formation of free radicals, and to counteract the damage of DNA, lipids and proteins. Luteolin (Lut), a common flavonoid present in many plants, has strong antioxidant and pharmacological activities, including a memory-improving effect. It displays excellent radical scavenging and cytoprotective properties, particularly when tested in complex biological systems where it can interact with other antioxidants, such as vitamins.27 Lut displays specific antiinflammatory effects, which are only partly explained by its antioxidant capacities. The antiinflammatory activity of Lut includes activation of antioxidative enzymes, suppression of the nuclear factor (NF)-B pathway and inhibition of pro-inflammatory substances. In vivo, Lut reduces increased vascular permeability and is effective in animal models of CNS inflammation.28, 29
In our study, we aimed to investigate the capability of Co-ultraPEALut, based on association of
PEA with Lut in fixed doses of 10:1 in mass, to counteract the secondary damage produced after
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 5 of 49
5
TBI, by analyzing the neuroprotective properties of this new Co-ultramicronized combination
formulation.
Page 6 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 Materials and Methods Animals Male CD1 mice weighing 25–30 g were kept five per cage under a constant 12-h light/dark cycle, at room temperature (23 °C). Food and water were available ad libitum. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). Controlled cortical impact (CCI) experimental TBI Surgical anesthesia was induced by ketamine and xylazine (2.6 and 0.16 mg/kg body weight respectively) administered intraperitoneally (i.p.). Following endotracheal intubation, the animals were secured in a stereotaxic frame and ventilated mechanically. A dab of sterile ophthalmic ointment was placed on each eye to compensate for the decrease in lacrimation during anesthesia. Utilizing aseptic techniques, a midline scalp incision was made, and the skin and fascia were reflected to expose the skull. A craniotomy was made in the right hemisphere encompassing bregma and lambda and between the sagittal suture and the coronal ridge with a Micro motor hand piece and drill (UGO Basile S.R.L., Comerio VA, italy). The resulting bone flap was removed, and the craniotomy enlarged further with cranial rongeurs. A cortical contusion was produced on the exposed cortex using a controlled impactor device Impact OneTM Stereotaxic impactor for CCI (myNeurolab.com, Richmond). Briefly, the impacting shaft was extended, and the impact tip was centered and lowered over the craniotomy site until it touched the Dura mater. Then, the rod was retracted and the impact tip was advanced farther to produce a brain injury of moderate severity for mice (tip diameter, 4 mm; cortical contusion depth, 3 mm; impact velocity, 1.5 m/sec). The impact tip was wiped clean with sterile alcohol after each impact and cleaned/disinfected further with Cidex after surgery. Core temperature was maintained at 37 ± 0.5°C with a heating pad during surgery and recorded with a rectal probe. Immediately after injury, the skin incision was closed with
Page 7 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 nylon sutures, and 2% lidocaine jelly was applied to the lesion site to minimize any possible discomfort. Experimental groups TBI was induced in mice (n = 10 for group) by controlled cortical impactor. Immediately after injury, the skin incision was closed with nylon sutures, and 2% lidocaine jelly was applied to the lesion site to minimize any possible discomfort. Two sets of experiments were performed. The first set was to investigate the protective effects of PEA and Co-ultraPEALut treatment. All animals were randomized into 1 of 4 groups: (i) Sham + vehicle group: mice were subjected to the surgical procedures (anesthesia and craniotomy) except that the impact tip was not applied and vehicle was administered 1 h after craniotomy; (ii) Sham + Co-ultraPEALut: mice were subjected to the surgical procedures as the group above (anesthesia and craniotomy) except that the impact tip was not applied and Co-ultraPEALut (1 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after craniotomy; (iii) Sham + PEA: mice were subjected to the surgical procedures as the group above (anesthesia and craniotomy) except that the impact tip was not applied and PEA (10 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after craniotomy; (iv) TBI + vehicle: mice were subjected to CCI and vehicle was administered 1 h after TBI; (v) TBI + Co-ultraPEALut: mice were subjected to CCI and Co-ultraPEALut (1 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after TBI; (vi) TBI + PEA: mice were subjected to CCI and Co-ultraPEALut (10 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after TBI;
Page 8 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 Mice (n = 10 from each group for each parameters) were killed at 24 h after TBI in order to evaluate the various parameter. In a second set of experiments other 10 animals for each group were observed after TBI in order to evaluate the behavioral testing.
Co-ultramicronization process of PEA and Lut The Co-ultramicronization process was performed using jet mill equipment (Sturtevant Inc., 348 Circuit Street Hanover, MA, USA) with a chamber of 300 mm in diameter, operated with ‘spiral technology’ and driven by compressed air at 10 to12 bars. Crashing was determined by the high number of collisions that occurred among particles as a result of the high level of kinetic (not mechanical) energy. This process is effective not only in reducing product particle size, but also in modifying crystalline structure. Scanning electron microscopy showed an intimate intermixing of PEA and Lut, while analysis by differential scanning calorimetry and X-ray diffraction indicated transformation into a crystalline form different from the original two, definable as ‘a higher energy content form’. The composite showed the following particle size distribution: 96%
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1 Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury
*
*
Marika Cordaro 1, Daniela Impellizzeri 1, Irene Paterniti1, Giuseppe Bruschetta1, Rosalba Siracusa1, Daniela De Stefano2, Salvatore Cuzzocrea1,3, Emanuela Esposito1
1.
Department of Biological and Environmental Sciences, University of Messina, Messina
2.
Department of Experimental Pharmacology University of Naples Federico II, Naples
3.
Manchester Biomedical Research Centre, Manchester Royal Infirmary, School of Medicine, University of Manchester, UK
*the authors contributed equally to this work
Cuzzocrea Salvatore, Full Professor.email: [email protected] Esposito Emanuela, Researcher Assistant. email: [email protected] De Stefano Daniela, Researcher Assistant. email: [email protected] Paterniti Irene, Post Doc. email: [email protected] Impellizzeri Daniela, Post Doc. email: [email protected] Cordaro Marika, PhD Student. email: [email protected] Bruschetta Giuseppe, PhD Student. email: [email protected] Siracusa Rosalba, Resident Student. email [email protected]
Corresponding Author: Prof. Salvatore Cuzzocrea - Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres, 31 – 98166 Messina, Italy; Phone: +390906765208; email: [email protected]
Running Title: Co-ultraPEALut and traumatic brain injury
Page 2 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Abstract Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. In the present study, we performed a widely-used model of TBI to determine the neuroprotective propriety of palmitoylethanolamide (PEA) and the antioxidant effect of a flavonoid luteolin (Lut), given as a co-ultramicronized compound Co-ultraPEALut. We demonstrated that the treatment with Co-ultraPEALut resulted in a significant improvement of motor and cognitive recovery after controlled cortical impact (CCI), as well as markedly reducing lesion volumes. Moreover, our results revealed the ability of Co-ultraPEALut, to reduce brain trauma through modulation of NF-κB activation. In addition, treatment with Co-ultraPEALut significantly enhanced the post-TBI expression of the neuroprotective neurotrophins GDNF compared to vehicle. Co-ultraPEALut at the dose of 1 mg/kg, also modulated apoptosis, the release of cytokine and ROS, the activation of chymase, tryptase and nitrotyrosine and inhibited autophagy. Thus, our data demonstrated that Co-ultraPEALut at a lower dose compared to PEA alone, can exert neuroprotective effects and the combination of both could improve their ability to counteract the neurodegeneration and neuroinflammation induced by TBI.
Keywords Neuroprotection, palmitoylethanolamide, luteolin, inflammation, autophagy
Page 3 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 Introduction Traumatic brain injury (TBI) is a major public health problem and a leading cause of mortality and morbidity worldwide, particularly among the young.1,
2
The American Brain Injury Association
describes TBI as “an alteration in brain function, or other evidence of brain pathology. caused by an external force”.3 TBI is a highly complex disorder that includes varying degrees of contusion, diffuse axonal injury, haemorrhage and hypoxia, defined primary injury.1, 4 Collectively, these effects induce biochemical and metabolic changes that lead to progressive tissue damage and associated cell death, called second injury.5 TBI induces a strong inflammatory response characterized by the recruitment of peripheral leukocytes and enhanced chymase, tryptase as well as nitrotyrosine activation into the cerebral parenchyma.6-8 Moreover, activated microglia migrates to injured sites and releases cytokines, chemotactic cytokines, reactive oxygen species (ROS), nitric oxide, proteases and other factors with cytotoxic effects, which may in turn exacerbate neuronal death.9, 10 Also, a close relationship exists between the degree of oxidative stress and the pathogenesis of TBI.11 Enhanced production of ROS and reactive nitrogen species (RNS) causes oxidative/nitrosative stress
leading to damage in
lipids,12, 13 proteins and nucleic acids.14, 15 Additionally, Lai et colleagues, have demonstrated that oxidative stress contributes to overall autophagy in mice after TBI and that this pathway was partially inhibited by an antioxidant.16 Autophagy, from Greek “self-eating” is a major protein degradation system that targets primarily long-lived cytoplasmic proteins.17 Autophagy involves a dynamic subcellular membrane rearrangement whereby cytoplasmic proteins and organelles are sequestered in double-membrane vesicular structures (autophagosomes) that fuse with the lysosome or vacuole, where the material is catabolically degraded, recycled and utilized as an energy source.18 Several key molecular components participate in the initiation, progression, and completion of autophagy, like the mammalian target of rapamycin (mTOR) that inhibits autophagy whereas Beclin 1 and LC3 promote it.
Page 4 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 In recent years, the pathophysiology of TBI has been a focus of extensive studies; animal models have been proved to be important tools in this field, and are employed to investigate the mechanisms of primary and secondary injury. Evaluation of improvement in neurological function using a clinically relevant TBI model such as controlled cortical impact (CCI) establishes efficacy and clinical relevance of an experimental therapy.8 In the CCI model, the injury to the brain initially presents as necrotic cell death in the underlying tissue and white matter axonal injury, both reminiscent of the clinical TBI pathology, but also followed by apoptotic cell death in surrounding tissue due to multiple subsequent events such as edema, ischemia, excitotoxicity and altered gene expression.19 Our previous works clearly demonstrated that treatment with palmitoylethanolamide (PEA) at 10 mg/kg significantly reduced the inflammation process associated with experimental spinal cord injury (SCI) 20 and TBI mice models.8 PEA is abundant in the central nervous system (CNS), and it is conspicuously produced by glial cells.21-23 It has been shown to inhibit peripheral inflammation and mast cell degranulation
24
, as
well as exert antinociceptive effects in rats and mice.25, 26 However, PEA lacks a direct antioxidant capacity to prevent the formation of free radicals, and to counteract the damage of DNA, lipids and proteins. Luteolin (Lut), a common flavonoid present in many plants, has strong antioxidant and pharmacological activities, including a memory-improving effect. It displays excellent radical scavenging and cytoprotective properties, particularly when tested in complex biological systems where it can interact with other antioxidants, such as vitamins.27 Lut displays specific antiinflammatory effects, which are only partly explained by its antioxidant capacities. The antiinflammatory activity of Lut includes activation of antioxidative enzymes, suppression of the nuclear factor (NF)-B pathway and inhibition of pro-inflammatory substances. In vivo, Lut reduces increased vascular permeability and is effective in animal models of CNS inflammation.28, 29
In our study, we aimed to investigate the capability of Co-ultraPEALut, based on association of
PEA with Lut in fixed doses of 10:1 in mass, to counteract the secondary damage produced after
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 5 of 49
5
TBI, by analyzing the neuroprotective properties of this new Co-ultramicronized combination
formulation.
Page 6 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 Materials and Methods Animals Male CD1 mice weighing 25–30 g were kept five per cage under a constant 12-h light/dark cycle, at room temperature (23 °C). Food and water were available ad libitum. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). Controlled cortical impact (CCI) experimental TBI Surgical anesthesia was induced by ketamine and xylazine (2.6 and 0.16 mg/kg body weight respectively) administered intraperitoneally (i.p.). Following endotracheal intubation, the animals were secured in a stereotaxic frame and ventilated mechanically. A dab of sterile ophthalmic ointment was placed on each eye to compensate for the decrease in lacrimation during anesthesia. Utilizing aseptic techniques, a midline scalp incision was made, and the skin and fascia were reflected to expose the skull. A craniotomy was made in the right hemisphere encompassing bregma and lambda and between the sagittal suture and the coronal ridge with a Micro motor hand piece and drill (UGO Basile S.R.L., Comerio VA, italy). The resulting bone flap was removed, and the craniotomy enlarged further with cranial rongeurs. A cortical contusion was produced on the exposed cortex using a controlled impactor device Impact OneTM Stereotaxic impactor for CCI (myNeurolab.com, Richmond). Briefly, the impacting shaft was extended, and the impact tip was centered and lowered over the craniotomy site until it touched the Dura mater. Then, the rod was retracted and the impact tip was advanced farther to produce a brain injury of moderate severity for mice (tip diameter, 4 mm; cortical contusion depth, 3 mm; impact velocity, 1.5 m/sec). The impact tip was wiped clean with sterile alcohol after each impact and cleaned/disinfected further with Cidex after surgery. Core temperature was maintained at 37 ± 0.5°C with a heating pad during surgery and recorded with a rectal probe. Immediately after injury, the skin incision was closed with
Page 7 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 nylon sutures, and 2% lidocaine jelly was applied to the lesion site to minimize any possible discomfort. Experimental groups TBI was induced in mice (n = 10 for group) by controlled cortical impactor. Immediately after injury, the skin incision was closed with nylon sutures, and 2% lidocaine jelly was applied to the lesion site to minimize any possible discomfort. Two sets of experiments were performed. The first set was to investigate the protective effects of PEA and Co-ultraPEALut treatment. All animals were randomized into 1 of 4 groups: (i) Sham + vehicle group: mice were subjected to the surgical procedures (anesthesia and craniotomy) except that the impact tip was not applied and vehicle was administered 1 h after craniotomy; (ii) Sham + Co-ultraPEALut: mice were subjected to the surgical procedures as the group above (anesthesia and craniotomy) except that the impact tip was not applied and Co-ultraPEALut (1 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after craniotomy; (iii) Sham + PEA: mice were subjected to the surgical procedures as the group above (anesthesia and craniotomy) except that the impact tip was not applied and PEA (10 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after craniotomy; (iv) TBI + vehicle: mice were subjected to CCI and vehicle was administered 1 h after TBI; (v) TBI + Co-ultraPEALut: mice were subjected to CCI and Co-ultraPEALut (1 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after TBI; (vi) TBI + PEA: mice were subjected to CCI and Co-ultraPEALut (10 mg/kg body weight, soluble 10% ethanol, i.p.) was administered 1 h after TBI;
Page 8 of 49
Journal of Neurotrauma Neuroprotective effects of Co-ultraPEALut on secondary inflammatory process and autophagy involved in traumatic brain injury (doi: 10.1089/neu.2014.3460) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 Mice (n = 10 from each group for each parameters) were killed at 24 h after TBI in order to evaluate the various parameter. In a second set of experiments other 10 animals for each group were observed after TBI in order to evaluate the behavioral testing.
Co-ultramicronization process of PEA and Lut The Co-ultramicronization process was performed using jet mill equipment (Sturtevant Inc., 348 Circuit Street Hanover, MA, USA) with a chamber of 300 mm in diameter, operated with ‘spiral technology’ and driven by compressed air at 10 to12 bars. Crashing was determined by the high number of collisions that occurred among particles as a result of the high level of kinetic (not mechanical) energy. This process is effective not only in reducing product particle size, but also in modifying crystalline structure. Scanning electron microscopy showed an intimate intermixing of PEA and Lut, while analysis by differential scanning calorimetry and X-ray diffraction indicated transformation into a crystalline form different from the original two, definable as ‘a higher energy content form’. The composite showed the following particle size distribution: 96%
