Cornelius K. Donat

Time-dependent alterations of cholinergic markers after experimental traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability. Cognitive deficits are believed to be connected with impairments of the cholinergic system. The present study was conducted to evaluate the cholinergic system in a model of focal brain injury with special attention to the time course of posttraumatic events in critical brain regions. Three groups of male Sprague-Dawley rats (post-TBI survival time: 2 h, 24 h and 72 h) were subjected to sham-operation (control) or controlled cortical impact injury. Receptor densities were determined on frozen ipsilateral sagittal brain sections with [(3)H]epibatidine (nicotinic acetylcholine receptors) and [(3)H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was evaluated with (-)[(3)H]vesamicol. Compared to control, vAChT was lowered (up to 50%) at each time point after trauma, with reductions in olfactory tubercle, basal forebrain, motor cortex, putamen, thalamic and hypothalamic areas and the gigantocellular reticular nucleus. Time-dependent reductions of about 20% of nAChR-density in the thalamus, hypothalamus, olfactory tubercle, gigantocellular reticular nucleus and motor cortex were observed post-TBI at 24 and 72 h. The same brain regions showed reductions of mAChR at 24 and 72 h after trauma with additional decreases in the corpus callosum, basal forebrain and anterior olfactory nucleus. In conclusion, cholinergic markers showed significant time-dependent impairments after TBI. Considering the role of the cholinergic system for cognitive processes in the brain, it seems likely that these impairments contribute to clinically relevant cognitive deficits.

Alterations of acetylcholinesterase activity after traumatic brain injury in rats

Objective: The cholinergic system is highly vulnerable to traumatic brain injury (TBI). However, limited information is available to what extent the degrading enzyme acetylcholinesterase (AChE) is involved. The present study addresses this question. Method: Thirty-six anaesthetized Sprague-Dawley rats were subjected to sham operation or to TBI using controlled cortical impact (CCI). The AChE activity was histochemically determined in frozen brain slices at 2, 24 and 72 hours after TBI. Results: High enzyme activity was observed in regions rich in cholinergic innervation such as the olfactory tubercle, basal forebrain, putamen and superior colliculi. Low activity was found in the cortex, cerebellum and particularly in the white matter. A decrease of AchE activity (20–35%) was found in the hippocampus and hypothalamus already at 2 hours after TBI. An increase of ∼30% was found in the basal forebrain at 2 and 24 hours. No changes occurred at 72 hours. Conclusion: The findings are consistent with impairment of the cholinergic neurotransmission after TBI and suggest the involvement of the AChE in short-term regulatory mechanisms.

Radiofluorination and biological evaluation of N-aryl-oxadiazolyl-propionamides as potential radioligands for PET imaging of cannabinoid CB2 receptors

Background The level of expression of cannabinoid receptor type 2 (CB2R) in healthy and diseased brain has not been fully elucidated. Therefore, there is a growing interest to assess the regional expression of CB2R in the brain. Positron emission tomography (PET) is an imaging technique, which allows quantitative monitoring of very low amounts of radiolabelled compounds in living organisms at high temporal and spatial resolution and, thus, has been widely used as a diagnostic tool in nuclear medicine. Here, we report on the radiofluorination of N-aryl-oxadiazolyl-propionamides at two different positions in the lead structure and on the biological evaluation of the potential of the two tracers [18F]1 and [18F]2 as CB2 receptor PET imaging agents. Results High binding affinity and specificity towards CB2 receptors of the lead structure remained unaffected by the structural changes such as the insertion of the aliphatic and aromatic fluorine in the selected labelling sites of 1 and 2. Aliphatic and aromatic radiofluorinations were optimized, and [18F]1 and [18F]2 were achieved in radiochemical yields of ≥30% with radiochemical purities of ≥98% and specific activities of 250 to 450 GBq/μmol. Organ distribution studies in female CD1 mice revealed that both radiotracers cross the blood–brain barrier (BBB) but undergo strong peripheral metabolism. At 30 min after injection, unmetabolized [18F]1 and [18F]2 accounted for 60% and 2% as well as 68% and 88% of the total activity in the plasma and brain, respectively. The main radiometabolite of [18F]2 could be identified as the free acid [18F]10, which has no affinity towards the CB1 and CB2 receptors but can cross the BBB. Conclusions N-aryl-oxadiazolyl-propionamides can successfully be radiolabelled with 18F at different positions. Fluorine substitution at these positions did not affect affinity and specificity towards CB2R. Despite a promising in vitro behavior, a rather rapid peripheral metabolism of [18F]1 and [18F]2 in mice and the generation of brain permeable radiometabolites hamper the application of these radiotracers in vivo. However, it is expected that future synthetic modification aiming at a replacement of metabolically susceptible structural elements of [18F]1 and [18F]2 will help to elucidate the potential of this class of compounds for CB2R PET studies.

Distinctive In Vivo Kinetics of the New σ1 Receptor Ligands (R)-(+)- and (S)-(–)-18F-Fluspidine in Porcine Brain

Because of their involvement in growth and survival signaling cascades, the σ1 receptors (σ1Rs) represent a novel target for the treatment of cancer and several brain diseases such as depression and neurodegeneration. From a series of σ1R-specific 18F-fluoroalkylated spirocyclic piperidines, we have chosen 18F-fluspidine for detailed investigation of the in vivo kinetics of the (R)-(+)- and (S)-(–)-enantiomers to identify their potential for imaging in humans. Methods: Enantiopure tosylate precursors for radiolabeling were obtained using chiral preparative high-performance liquid chromatography and used for radiosynthesis of both 18F-fluspidine enantiomers by nucleophilic substitution with K-18F-F-Kryptofix 222-carbonate complex in a synthesis module. Brain pharmacokinetics were investigated by dynamic PET studies in piglets under baseline and blocking conditions using the highly selective σ1R agonist SA4503. Standardized uptake values (SUVs) were calculated for 24 MR-defined brain regions. Total distribution volume (VT) and binding potentials (k3′/k4) of (S)-(–)- and (R)-(+)-18F-fluspidine were estimated. Furthermore, VT values were estimated by graphical analysis using Logan plots. Results: The (S)- and (R)-tosylates were obtained in excellent enantiomeric purities (>98% and >96% enantiomeric excess, respectively). (S)-(–)- and (R)-(+)-18F-fluspidine were synthesized within approximately 70 min (radiochemical yield, 35%–45%; specific activity, 650–870 GBq/μmol; radiochemical purity, >99%). Both radiotracers displayed different brain uptake kinetics. Although the initial brain uptake was similar, the SUV at the end of the study differed significantly (P < 0.05), with (R)-(+)-18F-fluspidine showing about 60%–150% higher values. Administration of SA4503 reduced SUV almost equally for both radiotracers by approximately 65%. Furthermore, k3′ was significantly decreased under blocking conditions in almost all regions ((S)-(–)-18F-fluspidine, −90%–95%; (R)-(+)-18F-fluspidine, −70%–90%) whereas effects on k4 differed according to the particular brain region. VT estimated by both graphical analysis using Logan plots and full nonlinear kinetic analysis revealed significant inhibition for both radiotracers under blocking conditions. Conclusion: Both (S)-(–)- and (R)-(+)-18F-fluspidine appear to be suitable for σ1R imaging in humans. The different pharmacokinetics of (S)-(–)-18F-fluspidine and (R)-(+)-18F-fluspidine may have the potential for application in the diagnostics of different pathologic conditions.

Microglial Activation in Traumatic Brain Injury

Microglia have a variety of functions in the brain, including synaptic pruning, CNS repair and mediating the immune response against peripheral infection. Microglia rapidly become activated in response to CNS damage. Depending on the nature of the stimulus, microglia can take a number of activation states, which correspond to altered microglia morphology, gene expression and function. It has been reported that early microglia activation following traumatic brain injury (TBI) may contribute to the restoration of homeostasis in the brain. On the other hand, if they remain chronically activated, such cells display a classically activated phenotype, releasing pro-inflammatory molecules, resulting in further tissue damage and contributing potentially to neurodegeneration. However, new evidence suggests that this classification is over-simplistic and the balance of activation states can vary at different points. In this article, we review the role of microglia in TBI, analyzing their distribution, morphology and functional phenotype over time in animal models and in humans. Animal studies have allowed genetic and pharmacological manipulations of microglia activation, in order to define their role. In addition, we describe investigations on the in vivo imaging of microglia using translocator protein (TSPO) PET and autoradiography, showing that microglial activation can occur in regions far remote from sites of focal injuries, in humans and animal models of TBI. Finally, we outline some novel potential therapeutic approaches that prime microglia/macrophages toward the beneficial restorative microglial phenotype after TBI.

Effect of leukotriene inhibitors on evolution of experimental brain contusions

C. Voigt, C. K. Donat, W. Hartig, A. Förschler, M. Skardelly, D. Stichtenoth, T. Arendt, J. Meixensberger and M. U. Schuhmann (2012) Neuropathology and Applied Neurobiology38, 354–366

Effects of lateral fluid percussion injury on cholinergic markers in the newborn piglet brain

Traumatic brain injury is a leading cause of death and disability in children. Studies using adult animal models showed alterations of the central cholinergic neurotransmission as a result of trauma. However, there is a lack of knowledge about consequences of brain trauma on cholinergic function in the immature brain. It is hypothesized that trauma affects the relative acetylcholine esterase activity and causes a loss of cholinergic neurons in the immature brain.

Development of a Novel Nonpeptidic 18F-Labeled Radiotracer for in Vivo Imaging of Oxytocin Receptors with Positron Emission Tomography

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

Impact of 5-lipoxygenase inhibitors on the spatiotemporal distribution of inflammatory cells and neuronal COX-2 expression following experimental traumatic brain injury in rats.

The inflammatory response following traumatic brain injury (TBI) contributes to neuronal death with poor outcome. Although anti-inflammatory strategies were beneficial in the experimental TBI, clinical translations mostly failed, probably caused by the complexity of involved cells and mediators. We recently showed in a rat model of controlled cortical impact (CCI) that leukotriene inhibitors (LIs) attenuate contusion growth and improve neuronal survival. This study focuses on spatiotemporal characteristics of macrophages and granulocytes, typically involved in inflammatory processes, and neuronal COX-2 expression. Effects of treatment with LIs (Boscari/MK-886), started prior trauma, were evaluated by quantifying CD68(+), CD43(+) and COX-2(+) cells 24h and 72 h post-CCI in the parietal cortex (PC), CA3 region, dentate gyrus (DG) and visual/auditory cortex (v/aC). Correlations were applied to identify intercellular relationships. At 24h, untreated animals showed granulocyte invasion in all regions, decreasing towards 72 h. Macrophages increased from 24h to 72 h post-CCI in PC and v/aC. COX-2(+) neurones showed no temporal changes, except of an increase in the CA3 region at 72 h. Treatment reduced granulocytes at 24h in the pericontusional zone and hippocampus, and macrophages at 72 h in the PC and v/aC. COX-2 expression remained unaffected by LIs, except of time-specific changes in the DG (increase/decrease at 24/72 h). Interrelations confirmed concomitant cellular reactions beyond the initial trauma site. In conclusion, LIs attenuated the cellular inflammatory response following CCI. Future studies have to clarify region-specific effects and explore the potential of a clinically more relevant therapeutic approach applying LIs after CCI.

Evaluation of the Enantiomer Specific Biokinetics and Radiation Doses of [18F]Fluspidine—A New Tracer in Clinical Translation for Imaging of σ1 Receptors

The enantiomers of [18F]fluspidine, recently developed for imaging of σ1 receptors, possess distinct pharmacokinetics facilitating their use in different clinical settings. To support their translational potential, we estimated the human radiation dose of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine from ex vivo biodistribution and PET/MRI data in mice after extrapolation to the human scale. In addition, we validated the preclinical results by performing a first-in-human PET/CT study using (S)-(−)-[18F]fluspidine. Based on the respective time-activity curves, we calculated using OLINDA the particular organ doses (ODs) and effective doses (EDs). The ED values of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine differed significantly with image-derived values obtained in mice with 12.9 μSv/MBq and 14.0 μSv/MBq (p < 0.025), respectively. A comparable ratio was estimated from the biodistribution data. In the human study, the ED of (S)-(−)-[18F]fluspidine was calculated as 21.0 μSv/MBq. Altogether, the ED values for both [18F]fluspidine enantiomers determined from the preclinical studies are comparable with other 18F-labeled PET imaging agents. In addition, the first-in-human study confirmed that the radiation risk of (S)-(−)-[18F]fluspidine imaging is within acceptable limits. However, as already shown for other PET tracers, the actual ED of (S)-(−)-[18F]fluspidine in humans was underestimated by preclinical imaging which needs to be considered in other first-in-human studies.