PET Imaging of Peripheral Benzodiazepine Receptor Standard Uptake Value Increases After Controlled Cortical Impact, a Rodent Model of Traumatic Brain Injury

Traumatic brain injury (TBI) is a chronic, life threatening injury for which few effective interventions are available. Evidence in animal models suggests un-checked immune activation may contribute to the pathophysiology. Changes in regional density of active brain microglia can be quantified in vivo with positron emission topography (PET) with the relatively selective radiotracer, peripheral benzodiazepine receptor 28 (11 C-PBR28). Phenotypic assessment (activated vs resting) can subsequently be assessed (ex vivo) using morphological techniques. To elucidate the mechanistic contribution of immune cells in due to TBI, we employed a hybrid approach involving both in vivo (11 C-PBR28 PET) and ex vivo (morphology) to elucidate the role of immune cells in a controlled cortical impact (CCI), a rodent model for TBI. Density of activated brain microglia/macrophages was quantified 120 hours after injury using the standardized uptake value (SUV) approach. Ex vivo morphological analysis from specific brain regions using IBA-1 antibodies differentiated ramified (resting) from amoeboid (activated) immune cells. Additional immunostaining of PBRs facilitated co-localization of PBRs with IBA-1 staining to further validate PET data. Injured animals displayed greater PBR28suv when compared to sham animals. Immunohistochemistry demonstrated elevated density of amoeboid microglia/macrophages in the ipsilateral dentate gyrus, corpus callosum, thalami and injury penumbra of injured animals compared to sham animals. PBR co-stained with amoeboid microglia/macrophages in the injury penumbra and not with astrocytes. These data suggest the technologies evaluated may serve as bio-signatures of neuroinflammation following severe brain injury in small animals, potentially enabling in vivo tracking of neuroinflammation following TBI and cellular-based therapies.

Benzodiazepine Receptors in the Periphery

The benzodiazepines are almost universally thought to produce one and only one pharmacologic effect: positive allosteric modulation of GABAA receptors located in the brain. This results in an increased Cl−ion influx, greater negative transmembrane potential difference, and neurons that are less likely to fire in response to anxiety-producing stimulation. Unfortunately, the simplicity and success of this mono-target belief has distracted researchers and clinicians from studying and appreciating their other pharmacology. A glaring example is the general lack of awareness of the peripheral benzodiazepine receptor. The peripheral benzodiazepine receptor alters mitochondrial function (energy supply), cholesterol transport, and immune function. A patient who is on long-term benzodiazepine therapy (or withdrawing from them) will have these sites affected, just as are the sites located in the brain. One can easily imagine that the adverse effects associated with the peripheral sites would be fundamental, varied, and potentially profound—involving lack of energy, altered cholesterol metabolism, and aberrant immune function.

The ligands of translocator protein: design and biological properties

: In 2020, it is already 43 years since Braestrup and Squires discovered 18 kDa translocator protein (TSPO), known until 2006 as "peripheral benzodiazepine receptor". During this time the functions of this receptor which is located on the outer membrane of mitochondria were studied in detail. One of the key functions of TSPO is the transfer of cholesterol from the outer to the inner mitochondrial membrane, which is the limiting stage in the synthesis of neurosteroids. TSPO is also involved in the transport of porphyrins, mitochondrial respiration, the opening of mitochondrial pores, apoptosis and cell proliferation. This review presents current information on the structure of TSPO, the mechanism of its participation in neurosteroidogenesis, as well as endogenous and synthetic TSPO ligands. Particular emphasis is placed on the analysis of approaches to the design of synthetic ligands and their neuropsychotropic activity in vitro and in vivo. The presented review demonstrates the promise of constructing new neuropsychotropic drugs in the series of TSPO ligands.

PET Imaging of Peripheral Benzodiazepine Receptor Standard Uptake Value Increases After Controlled Cortical Impact, a Rodent Model of Traumatic Brain Injury.

Background: Traumatic brain injury (TBI) disrupts the complex arrangement of neuronal and glial cells. As a result of TBI there is activation of microglia. Activated microglia after injury can be measured in vivo by using positron emission topography (PET) ligand peripheral benzodiazepine receptor (PBR28) and their phenotypes (activated vs resting) can be assessed (ex vivo) using morphology. This study aims to utilize in vivo (PET) and ex vivo (morphology) to assess the changes in microglia after a controlled cortical impact (CCI), a rodent model for TBI.Methods: Male Sprague Dawley rats underwent a sham injury or severe CCI. Microglia activation was assessed 120 hours after the injury by PET/CT imaging using the radioligand [11C] PBR-28. Standardized uptake values (PBR28suv) were calculated over the duration of the scan and mean values were compared. In order to verify in vivo results, ex vivo morphological analysis [ramified (resting) or amoeboid-shaped (activated)] was performed (dentate gyrus, corpus callosum and thalamus) with the antibody IBA-1. To further conclude that PBR is a marker for activated microglia after CCI, we examined co-staining of PBR with microglia and astrocytes.Results: In vivo and ex vivo results were complementary. Injured animals displayed greater PBR28suv when compared to sham animals. Immunohistochemistry demonstrated elevated numbers of activated microglia in the ipsilateral dentate gyrus, corpus callosum and thalami of injured animals compared to sham animals. Additionally, PBR co-stained with microglia and not astrocytes.Conclusion: CCI, a rodent model of TBI resulted in a significant increase in PBR28suv due to injury. Similarly, morphological analysis demonstrated a significant increase in amoeboid-shaped (activated) microglia. These results serve as a surrogate marker for increased neuroinflammation in the brains of severely injured animals. PBR28suv can serve as an in vivo tracking system for monitoring neuroinflammation following TBI and cellular therapies.

Midazolam and Dexmedetomidine Affect Neuroglioma and Lung Carcinoma Cell Biology In Vitro and In Vivo

Editor’s PerspectiveWhat We Already Know about This TopicWhat This Article Tells Us That Is NewBackgroundSeveral factors within the perioperative period may influence postoperative metastatic spread. Dexmedetomidine and midazolam are widely used general anesthetics during surgery. The authors assessed their effects on human lung carcinoma (A549) and neuroglioma (H4) cell lines in vitro and in vivo.MethodsCell proliferation and migration were measured after dexmedetomidine (0.001 to 10 nM) or midazolam (0.01 to 400 μM) treatment. Expression of cell cycle and apoptosis markers were assessed by immunofluorescence. Mitochondrial membrane potential and reactive oxygen species were measured by JC-1 staining and flow cytometry. Antagonists atipamezole and flumazenil were used to study anesthetic mechanisms of action. Tumor burden after anesthetic treatment was investigated with a mouse xenograft model of lung carcinoma.ResultsDexmedetomidine (1 nM) promoted cell proliferation (2.9-fold in A549 and 2-fold in H4 cells vs. vehicle, P < 0.0001; n = 6), migration (2.2-fold in A549 and 1.9-fold in H4 cells vs. vehicle, P < 0.0001; n = 6), and upregulated antiapoptotic proteins in vitro. In contrast, midazolam (400 μM) suppressed cancer cell migration (2.6-fold in A549 cells, P < 0.0001; n = 4), induced apoptosis via the intrinsic mitochondrial pathway, decreased mitochondrial membrane potential, and increased reactive oxygen species expression in vitro—effects partly attributable to peripheral benzodiazepine receptor activation. Furthermore, midazolam significantly reduced tumor burden in mice (1.7-fold vs. control; P < 0.05; n = 6 per group).ConclusionsMidazolam possesses antitumorigenic properties partly mediated by the peripheral benzodiazepine receptor, whereas dexmedetomidine promotes cancer cell survival through signaling via the α2-adrenoceptor in lung carcinoma and neuroglioma cells.