Brain activation induced by voluntary alcohol and saccharin drinking in rats assessed with manganese-enhanced magnetic resonance imaging.

The neuroanatomical and neurochemical basis of alcohol reward has been studied extensively, but global alterations of neural activity in reward circuits during chronic alcohol use remain poorly described. Here, we measured brain activity changes produced by long-term voluntary alcohol drinking in the alcohol-preferring AA (Alko alcohol) rats using manganese-enhanced magnetic resonance imaging (MEMRI). MEMRI is based on the ability of paramagnetic manganese ions to accumulate in excitable neurons and thereby enhance the T1-weighted signal in activated brain areas. Following 6 weeks of voluntary alcohol drinking, AA rats were allowed to drink alcohol for an additional week, during which they were administered manganese chloride (MnCl2 ) with subcutaneous osmotic minipumps before MEMRI. A second group with an identical alcohol drinking history received MnCl2 during the abstinence week following alcohol drinking. For comparing alcohol with a natural reinforcer, MEMRI was also performed in saccharin-drinking rats. A water-drinking group receiving MnCl2 served as a control. We found that alcohol drinking increased brain activity extensively in cortical and subcortical areas, including the mesocorticolimbic and nigrostriatal dopamine pathways and their afferents. Remarkably similar activation maps were seen after saccharin ingestion. Particularly in the prelimbic cortex, ventral hippocampus and subthalamic nucleus, activation persisted into early abstinence. These data show that voluntary alcohol recruits an extensive network that includes the ascending dopamine systems and their afferent connections, and that this network is largely shared with saccharin reward. The regions displaying persistent alterations after alcohol drinking could participate in brain networks underlying alcohol seeking and relapse.

Intraluminal cell transplantation prevents growth and rupture in a model of rupture-prone saccular aneurysms.

BACKGROUND AND PURPOSE: Aneurysm occlusion by intraluminal thrombus formation is the desired effect of all endovascular treatments. Intraluminal thrombus may, however, recanalize and be absorbed, unless it is infiltrated by cells that turn it into fibrous tissue (neointima). Because ruptured aneurysm walls are characterized by loss of smooth muscle cells, we assessed the impact of mural cell loss on wall remodeling of thrombosed aneurysms and investigated whether neointima formation could be enhanced by direct transplantation of cells into the thrombus. METHODS: Sidewall aneurysms were microsurgically created in rats (n=81). Certain aneurysms were decellularized. Thrombosis was induced using direct injection of a fibrin polymer into the aneurysm. CM-Dil-labeled smooth muscle cells were injected into 25 of 46 fibrin embolized aneurysms. Recanalization and aneurysm growth were monitored with magnetic resonance angiography. Endoscopy, optical projection tomography, histology, and immunohistochemistry were used to study the fate of transplanted cells, thrombus organization, and neointima formation. RESULTS: Decellularized embolized aneurysms demonstrated higher angiographic recurrence compared with decellularized embolized aneurysms with transplanted cells (P=0.037). Local cell replacement at the time of thrombosis resulted in better histological neointima formation than both nondecellularized embolized aneurysms (P<0.001) and decellularized embolized aneurysms (P=0.002). Aneurysm growth and rupture were observed exclusively in decellularized embolized aneurysms. CONCLUSIONS: Lack of smooth muscle cells in the aneurysm wall promotes wall degradation, aneurysm growth and rupture, even if the aneurysm is occluded by luminal thrombus. Transplantation of smooth muscle cells into the luminal thrombus can reduce this degenerative remodeling.

Loss of mural cells leads to wall degeneration, aneurysm growth, and eventual rupture in a rat aneurysm model.

BACKGROUND AND PURPOSE: The biological mechanisms predisposing intracranial saccular aneurysms to growth and rupture are not yet fully understood. Mural cell loss is a histological hallmark of ruptured cerebral aneurysms. It remains unclear whether mural cell loss predisposes to aneurysm growth and eventual rupture. METHODS: Sodium dodecyl sulfate decellularized and nondecellularized saccular aneurysm from syngeneic thoracic aortas were transplanted to the abdominal aorta of Wistar rats. Aneurysm patency and growth was followed up for 1 month with contrast-enhanced serial magnetic resonance angiographies. Endoscopy and histology of the aneurysms were used to assess the role of periadventitial environment, aneurysm wall, and thrombus remodeling. RESULTS: Nondecellularized aneurysms (n=12) showed a linear course of thrombosis and remained stable. Decellularized aneurysms (n=12) exhibited a heterogeneous pattern of thrombosis, thrombus recanalization, and growth. Three of the growing aneurysms (n=5) ruptured during the observation period. Growing and ruptured aneurysms demonstrated marked adventitial fibrosis and inflammation, complete wall disruption, and increased neutrophil accumulation in unorganized intraluminal thrombus. CONCLUSIONS: In the presented experimental setting, complete loss of mural cells acts as a driving force for aneurysm growth and rupture. The findings suggest that aneurysms missing mural cells are incapable to organize a luminal thrombus, leading to recanalization, increased inflammatory reaction, severe wall degeneration, and eventual rupture.

Donor simvastatin treatment abolishes rat cardiac allograft ischemia/reperfusion injury and chronic rejection through microvascular protection.

BACKGROUND: Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. METHODS AND RESULTS: Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell-endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury-induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-ß1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-ß1-induced microvascular endothelial-to-mesenchymal transition. CONCLUSIONS: Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

High-resolution TOF MR angiography at 4.7 Tesla for volumetric and morphologic evaluation of coiled aneurysm neck remnants in a rat model.

BACKGROUND: Detection of morphologic and volumetric changes in aneurysm necks is important when evaluating the effects of endovascular devices for aneurysm occlusion. PURPOSE: To optimize high-resolution 3D-TOF MRA at 4.7 T in order to achieve the best aneurysm-to-background contrast in experimental rat aneurysms, and to quantify the volume of the aneurysm neck by imaging. MATERIAL AND METHODS: Saccular aneurysms in the abdominal aorta of rats were coiled with platinum coils. Tissue relaxation times were measured, and used in a mathematical simulation. To optimize 3D-TOF angiography, imaging parameters were varied within the range obtained from the simulations. Tissue contrast and contrast-to-noise ratio were measured from MR images. TOF images were compared to conventional spin echo and gradient echo images and to endoscopic and histological analyses. RESULTS: Parameters yielding the best aneurysm-to-background contrast and contrast-to-noise ratio were determined. The agreement between the results from in vivo imaging and simulation was good. The optimized 3D-TOF MRA sequence (TR/TE/FA = 60 ms/6.6 ms/20°) had an isotropic voxel size of 117 µm, which enabled measurement of the aneurysm neck volume. CONCLUSION: High-resolution 3D-TOF angiography enables non-invasive quantification of changes in neck remnants of endovascularly coiled experimental aneurysms. In this model innovations like bioactive coils can be accurately tested for their efficacy for aneurysm occlusion.

Adenoviruses with an αvß integrin targeting moiety in the fiber shaft or the HI-loop increase tumor specificity without compromising antitumor efficacy in magnetic resonance imaging of colorectal cancer metastases.

BACKGROUND: Colorectal cancer is often a deadly disease and cannot be cured at metastatic stage. Oncolytic adenoviruses have been considered as a new therapeutic option for treatment of refractory disseminated cancers, including colorectal cancer. The safety data has been excellent but tumor transduction and antitumor efficacy especially in systemic administration needs to be improved. METHODS: Here, the utility of αvß integrin targeting moiety Arg-Gly-Asp (RGD) in the Lys-Lys-Thr-Lys (KKTK) domain of the fiber shaft or in the HI-loop of adenovirus serotype 5 for increased tumor targeting and antitumor efficacy was evaluated. To this end, novel spleen-to-liver metastatic colorectal cancer mouse model was used and the antitumor efficacy was evaluated with magnetic resonance imaging (MRI). RESULTS: Both modifications (RGD in the HI-loop or in the fiber shaft) increased gene transfer efficacy in colorectal cancer cell lines and improved tumor-to-normal ratio in systemic administration of the vector. CONCLUSIONS: Antitumor potency was not compromised with RGD modified viruses suggesting increased safety profile and tumor specificity.

Optimization of high-resolution USPIO magnetic resonance imaging at 4.7 T using novel phantom with minimal structural interference.

PURPOSE: To characterize the effect of ultrasmall superparamagnetic iron oxides (USPIOs) on magnetic resonance imaging (MRI) signal at 4.7 T, and to find the highest sensitivity pulse sequence for high-resolution USPIO MRI. MATERIALS AND METHODS: A novel phantom was constructed for optimization of sequence parameters for neuroradiological MR applications, and a wide range of dilutions of the USPIO ferumoxtran-10 was imaged using T(2)/T(1)-, T(1)-, T(2)-, T* (2)-, and PD-weighted sequences. The effect of varying sequence parameters was investigated using phantom measurements and simulations. RESULTS: The relaxivities r(1), r(2), and r*(2) of ferumoxtran-10 at 4.7 T (21°C) were 5.1, 82.2, and 148.4 mmol(-1) L s(-1), respectively. Gradient echo sequences produced superior susceptibility artifacts at high concentrations; susceptibility artifacts were seen down to a concentration of 137 nmol Fe/mL. A concentration of 17.5 µmol Fe/mL caused a signal void independently of sequence and parameters, and at concentrations ≤273 nmol Fe/mL no signal void was caused. Signal enhancement on T(1)-weighted imaging was seen only at concentrations 137-547 nmol Fe/mL. For the same effective echo time T(2)-weighted rapid acquisition with relaxation enhancement (RARE) yielded significantly higher contrast-to-noise ratio with RARE factor 16 than with RARE factor 8. CONCLUSION: At nanomolar concentrations of USPIO, steady-state free precession offers an alternative to T(2)- and T*(2)-weighted sequences. Optimum parameters depend highly on USPIO concentration.

A basic MRI anatomy of the rat brain in coronal sections for practical guidance to neuroscientists.

Identification of the brain structures in the magnetic resonance imaging (MRI) of the rat is very important for the experimental work of many neuroscientists. Our intention was to recognize most of the structures without overlapping the MRI sections with the histological templates. Three live rats were used for this study who were examined in a micro-MRI apparatus by performing T2-weighted sequences in serial brain sections. Most of the white matter structures were easily identified, e.g. the anterior commissure, corpus callosum with forceps minor and major, cingulum, external and internal capsules, fornix, stria medullaris and terminalis, cranial nerves, mammillothalamic tract, fasciculus retroflexus, medial and lateral lemniscus, posterior commissure, commissures of the superior and inferior colliculi, medial longitudinal fasciculus, and the cerebral peduncle. Large and small gray matter structures were recognized as well, for example, the anterior olfactory structures, nucleus accumbens, caudate putamen, claustrum, bed nucleus of the stria terminalis, pituitary gland, globus pallidus, amygdala, some midline and intralaminar thalamic nuclei, certain hypothalamic nuclei, hippocampal formation, pineal body, periaqueductal gray matter, lateral and medial geniculate bodies, superior and inferior colliculi, and cranial nerves nuclei. All in all, of the total 160 recognized brain structures, 77 were identified without using the corresponding histological atlases. We believe that our labeled MRI pictures could be an important way for quick orientation for evaluating the effects of the experimental work regarding the rat brain.

Occlusion of neck remnant in experimental rat aneurysms after treatment with platinum- or polyglycolic-polylactic acid-coated coils.

BACKGROUND: Neck remnants and aneurysm recurrences are marked limitations of endovascular treatment of cerebral artery aneurysms. We compared the evolution of neck remnants of experimental arterial rat aneurysms after treatment with either platinum- or PGLA-coated coils. METHODS: We created 20 standard-size aneurysms in the abdominal aortas of male Wistar rats. Aneurysms were embolized with either PGLA-coated coils or platinum coils, with care taken to leave a neck remnant. Neck remnant size and shape was closely monitored to detect progressive enlargement or occlusion. Using a 4.7 T MR scanner, we acquired high-resolution MR images 6 times during the 4-week follow-up. For quantitative measurements, we used a high-resolution 3D-TOF angiography sequence. Results were verified by endoscopy and histology. RESULTS: Aneurysms treated with PGLA coils showed, on average, a 12.9% reduction of neck remnant size (P = .044) and significant disappearance of dog ears, the blood-filled spaces between coils and aneurysm wall. The aneurysms treated with platinum coils lacked these changes. In endoscopy, neointima was found to cover both PGLA and platinum coils but was more often incomplete or translucent in the platinum group. In histology, thrombus organization and inflammatory cell infiltration were higher with PGLA. CONCLUSION: Use of PGLA-coated coils was followed by a moderate progressive reduction of the neck remnant size and a better angiographic outcome, seen as disappearance of dog ears during follow-up. The rat model proved to be suitable for comparison of different coil types.

Poststroke delivery of MANF promotes functional recovery in rats.

Stroke is the most common cause of adult disability in developed countries, largely because spontaneous recovery is often incomplete, and no pharmacological means to hasten the recovery exist. It was recently shown that mesencephalic astrocyte-derived neurotrophic factor (MANF) induces alternative or M2 activation of immune cells after retinal damage in both fruit fly and mouse and mediates retinal repair. Therefore, we set out to study whether poststroke MANF administration would enhance brain tissue repair and affect behavioral recovery of rats after cerebral ischemic injury. We used the distal middle cerebral artery occlusion (dMCAo) model of ischemia-reperfusion injury and administered MANF either as a recombinant protein or via adeno-associated viral (AAV) vector. We discovered that, when MANF was administered to the peri-infarct region 2 or 3 days after stroke, it promoted functional recovery of the animals without affecting the lesion volume. Further, AAV7-MANF treatment transiently increased the number of phagocytic macrophages in the subcortical peri-infarct regions. In addition, the analysis of knockout mice revealed the neuroprotective effects of endogenous MANF against ischemic injury, although endogenous MANF had no effect on immune cell-related gene expression. The beneficial effect of MANF treatment on the reversal of stroke-induced behavioral deficits implies that MANF-based therapies could be used for the repair of brain tissue after stroke.

Late radiation effects in the dog brain: correlation of MRI and histological changes.

PURPOSE: To determine the correlation between sequential changes in the brain of dogs after irradiation, as detected by magnetic resonance imaging (MRI), with the eventual appearance of histological lesions. Histology was performed 77-115 weeks after irradiation. MATERIALS AND METHODS: Groups of five beagle dogs were irradiated to the brain with single doses of 10, 12, 14 or 16 Gy of 6 MV photons, at the 100% iso-dose. Sequential MRIs were taken to detect changes in the brain for 77-115 weeks after irradiation. Dose-effect relationships were established for changes in the brain as detected by MRI, computerized tomography (CT), gross morphology and histology. The doses that caused a specified response in 50% of the animals (ED(50)+/-SE) were calculated from these dose-effect relationships for each endpoint. RESULTS: The ED50 values (+/-SE) for focal and diffuse changes on T2-weighted MR images were 11.0+/-1.1 and 10.8+/-0.9 Gy, respectively. The ED50 values (+/-SE) for contrast enhancement on T1-weighted MR images and on CT were 13.4+/-0.6 and 13.0+/-0.6 Gy, respectively. It was 11.4+/-0.6 Gy for any type of histological lesion (haemorrhage, reactive change or glial scar) 77-115 weeks after irradiation. For a macroscopic lesion the ED50 (+/-SE) value was 13.0+/-1.1 Gy. CONCLUSIONS: The presence of focal or diffuse changes on T2-weighted MR images was the best indicator for the eventual appearance of any type of histological lesion in the dog brain after irradiation with single doses of photons. The ED50 for any histological lesion did not differ significantly from the ED50 for a focal (P>0.35) or diffuse (P=0.3) change on T2-weighted MR images.

The role of diffusion- and perfusion-weighted magnetic resonance imaging in drug development for ischemic stroke: from laboratory to clinics.

Ischemic stroke is a major cause of mortality and morbidity in industrialized countries and is almost always caused by occlusion of a cerebral artery by a clot. As the reversibly injured brain tissue evolves into irreversible infarction within a short period of time after onset of ischemia, it is extremely important and urgent to reverse the serious consequences of brain ischemia in the hyperacute phase when the ischemic brain tissue is still salvageable. Numerous thrombolytic and potentially neuroprotective agents have been studied in stroke patients with little success as the only approved therapy is thrombolysis with recombinant tissue plasminogen activator (r-tPA) within 3 h of stroke onset in highly selected patients (approximately 5 to 10 % of all acute stroke patients). One major obstacle in the development of effective therapies for ischemic stroke has been the lack of versatile imaging techniques. New magnetic resonance imaging (MRI) modalities, specially diffusion- and perfusion-weighted MRI (DWI and PWI, respectively) have been used in experimental studies with great success for over a decade and now are gradually entering clinical use. DWI and PWI can detect brain ischemia in the early phase in its full extent thus ensuring a definite diagnosis, allowing for follow-up of the ischemic lesion size over time with good spatial and temporal resolution, demonstrating perfusion deficit and reperfusion and the existence and the extent of penumbra while only requiring a few minutes of imaging time. DWI and PWI do not just give us the correct diagnosis of ischemic stroke, but allow us to acquire in vivo lesion size before therapeutic regimen is started and monitor the therapeutic efficacy thereafter, thus overcoming the potential pretreatment bias. We used DWI and PWI to evaluate novel therapeutic approaches for ischemic stroke in numerous experimental studies and lately in humans. With DWI and PWI, we are able to determine the in vivo efficacy (or lack of efficacy) of new therapeutic regiments (both neuroprotective and thrombolytic agents, or combination therapies) in a rapid, safe, and reliable way and in a relatively small number of well-selected, well-defined, and homogeneous patients. This approach may, therefore, significantly accelerate the development of new remedies for stroke patients.

MnDPDP-enhanced magnetization transfer MR imaging: implications for effective liver imaging.

The benefit of combining magnetization transfer (MT) MR imaging technique with liver-specific contrast agent manganese dipyridoxyldiphosphate (MnDPDP) was assessed in our experimental investigation. The study was accomplished by imaging a phantom containing serial concentrations of MnDPDP in cross-linked bovine serum albumin (BSA) with various protein concentrations. A 0.1T clinical MR imager with different parameters for MT and conventional MR sequences were used. The combination of an offset frequency of 8 kHz and an amplitude of 25 microT produced nearly maximal MT effect for all protein samples either without MnDPDP or with different MnDPDP concentrations. With long TRs (TR > 200 ms) MT dramatically improved CNR in conjunction with MnDPDP. With short TRs, the gain in CNR with MT was negligible. However, long TRs with increased number of images are beneficial in liver imaging. We conclude that MT like preparation pulse is useful when paramagnetic contrast agents such as MnDPDP are employed.

AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

BACKGROUND: For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. NEW METHOD: We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). RESULTS: We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. COMPARISON WITH EXISTING METHODS: Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. CONCLUSIONS: We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process.

Visualization of luminal thrombosis and mural Iron accumulation in giant aneurysms with Ex vivo 4.7T magnetic resonance imaging.

BACKGROUND: Better diagnostic tools to identify rupture-prone saccular intracranial aneurysms (sIA) are needed. Inflammation and luminal thrombus associate with degeneration and rupture of the sIA wall. Iron-uptake has been detected in the inflammatory cells of the sIA wall and thrombus is the likely source of this iron. We investigated ex vivo the use of magnetic resonance imaging (MRI) to detect iron accumulation and luminal thrombus in giant sIAs. METHODS: Giant sIAs (n = 3) were acquired from microsurgical operations, fixed with formalin, embedded in agar and imaged at 4.7T. Samples were sectioned maintaining the orientation of the axial plane of MRI scans, and stained (hematoxylin-eosin and Prussian blue). RESULTS: All three giant sIAs showed a degenerated hypocellular wall with both mural and adventitial iron accumulation and displayed different degrees of luminal thrombus formation and thrombus organization. Signal intensity varied within the same sIA wall and associated with iron accumulation in all tested sequences. Wall areas with iron accumulation had significantly lower signal to noise ratio (SNR) compared with areas without iron accumulation (P = 0.002). Fresh and organizing thrombus differed in their MRI presentation and differed in signal intensity of the aneurysm wall (P = 0.027). CONCLUSION: MRI can detect ex vivo the accumulation of iron in giant sIA wall, as well as fresh and organizing luminal thrombus. These features have been previously associated with fragile, rupture-prone aneurysm wall. Further studies of iron accumulation as a marker of rupture-prone aneurysm wall are needed.

Evolution of intracerebral hemorrhage after intravenous tPA: reversal of harmful effects with mast cell stabilization.

Thrombolysis with tissue plasminogen activator (tPA) traditionally demands baseline imaging to rule out intracerebral hemorrhage (ICH), which causes delays in treatment. Preventing possible adverse effects of tPA on ICH would allow rapid on-site thrombolysis in patients with presumed acute ischemic stroke, reducing onset-to-treatment times. We examined how intravenous tPA alters ICH evolution during an extended follow-up, and how mast cell stabilization affects this process. Intracerebral hemorrhage was induced in rats by collagenase injection. Rats received either saline (n=10), tPA (n=13), tPA+low-dose cromoglycate (n=10), or tPA+high-dose cromoglycate (n=10). Magnetic resonance imaging was performed at 24, 48, and 72 hours after ICH induction, together with neurologic evaluations. During 72 hours of follow-up, tPA administration did not significantly increase hematoma volume (mean±s.d. 83.5±14.3 versus 66.7±14.7 µL; P=0.256) or hemispheric expansion (14.5±5.0 versus 11.5±5.0%; P=0.457) compared with saline. However, tPA-treated animals had worse neurologic outcomes (P<0.05), and mortality (8/13 versus 3/10). Combining tPA with high-dose cromoglycate mitigated hemispheric expansion (7.4±1.7 versus 14.5±5.0%; P=0.01), improved neurologic outcome (P<0.001) and decreased mortality (1/10; P<0.05) compared with tPA alone. Our results suggest tPA increases neurologic deficit in ICH, an effect that was abolished by concomitant mast cell stabilization. Further studies are needed to establish the clinical relevance of these findings.

Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

Prostatic acid phosphatase (PAP), the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG), but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP) in the brain by utilizing mice deficient in TMPAP (PAP-/- mice). Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

Long-term evolution of diffusion tensor indices after temporary experimental ischemic stroke in rats.

Diffusion tensor (DT) imaging measures the random molecular diffusion of water in vivo and provides information on the microstructure of tissue. Ischemic brain damage leads to tissue disorganization and structural lost. We aimed to evaluate these changes in a rat model of focal stroke from the hyperacute to chronic phase by utilizing several DT indices. Adult male Wistar rats, subjected to temporary focal cerebral ischemia by suture occlusion of the middle cerebral artery for 90min, and sham controls were serially imaged at 4.7Tesla. DT scans were collected repeatedly during the hyperacute (2 and 3.5h), acute (1, 2, and 3days), subacute (4, 7, and 14days), and chronic (4, 6, and 8weeks) phases. We measured the evolution of DT indices (mean diffusivity (MD), axial diffusivity (λ(║)), radial diffusivity (λ(┴)), and fractional anisotropy (FA)) in the cortex, subcortex, and corpus callosum of the ischemic hemisphere. In the hyperacute phase, MD, λ(║), and λ(┴) reduced with no change in FA. From the acute to subacute phase, MD, λ(║), and λ(┴) normalized and thereafter increased, whereas FA decreased in all the tissues. In the chronic phase, MD, λ(║), and λ(┴) continued to rise, whereas FA normalized in the corpus callosum and subcortex, but remained low in the cortex. We described structural tissue changes in ischemic rat brain longitudinally utilizing DT analysis. DT indices reveal different individual patterns reflecting different facades and phases of tissue injury. The use of several DT indices may improve accuracy in estimating the age of the brain injury and in detecting ongoing pathological events.

Optimized mouse model for the imaging of tumor metastasis upon experimental therapy.

Development of new cancer treatments focuses increasingly on the relation of cancer tissue with its microenvironment. A major obstacle for the development of new anti-cancer therapies has been the lack of relevant animal models that would reproduce all the events involved in disease progression from the early-stage primary tumor until the development of mature metastatic tissue. To this end, we have developed a readily imageable mouse model of colorectal cancer featuring highly reproducible formation of spontaneous liver metastases derived from intrasplenic primary tumors. We optimized several experimental variables, and found that the correct choice of cell line and the genetic background, as well as the age of the recipient mice, were critical for establishing a useful model system. Among a panel of colorectal cancer cell lines tested, the epithelial carcinoma HT29 line was found to be the most suitable in terms of producing homogeneous tumor growth and metastases. In our hands, SCID mice at the age of 125 days or older were the most suitable in supporting consistent HT29 tumor growth after splenic implantation followed by reproducible metastasis to the liver. A magnetic resonance imaging (MRI) protocol was optimized for use with this mouse model, and demonstrated to be a powerful method for analyzing the antitumor effects of an experimental therapy. Specifically, we used this system to with success to verify by MRI monitoring the efficacy of an intrasplenically administered oncolytic adenovirus therapy in reducing visceral tumor load and development of liver metastases. In summary, we have developed a highly optimized mouse model for liver metastasis of colorectal cancer, which allows detection of the tumor load at the whole body level and enables an accurate timing of therapeutic interventions to target different stages of cancer progression and metastatic development.

An optimized mouse model for transient ischemic attack.

Transient ischemic attacks (TIAs) are brief neurological deficits ofcerebrovascular origin that are followed by complete clinical recovery. Although a plethora of animal models exist for ischemic stroke, a verified TIA model is lacking. We aimed to optimize such a model in mice, investigating the impact of varying durations (from 2.5 to 20 minutes) of intraluminal middle cerebral artery occlusion (MCAo). Three conditions were required to mimic clinical TIA reliably: 1) an objective demonstration of occlusion and reperfusion (assessed by laser Doppler flowmetry); 2) no permanent neurological deficit (assessed by sensorimotor neurological evaluation); and 3) no lesion at 24 hours after reperfusion (assessed by magnetic resonance imaging [MRI]). We observed high incidences of MRI lesions with MCAo durations of 15 minutes or longer. In contrast, no permanent neurological deficits or MRI lesions were observed in animals with MCAo below or equal to 10 minutes. Middle cerebral artery occlusion of 12.5 minutes rarely induced MRI lesions, but histopathologic evaluation using routine and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining revealed minute ischemic changes even after 2.5-minute MCAo. Abundance of necrotic and apoptotic changes gradually increased with the duration of ischemia. These results indicate that 10 minutes or shorter focal cerebral ischemia proves a suitable mouse TIA model; in addition, they indicate that MRI-negative microscopic ischemic damage may occur with even a few minutes of arterial occlusion.