Jackeline Moraes Malheiros

Changes in Hippocampal Volume are Correlated with Cell Loss but Not with Seizure Frequency in Two Chronic Models of Temporal Lobe Epilepsy

Kainic acid (KA) or pilocarpine (PILO) have been used in rats to model human temporal lobe epilepsy (TLE) but the distribution and severity of structural lesions between these two models may differ. Magnetic resonance imaging (MRI) studies have used quantitative measurements of hippocampal T2 (T2HP) relaxation time and volume, but simultaneous comparative results have not been reported yet. The aim of this study was to compare the MRI T2HP and volume with histological data and frequency of seizures in both models. KA- and PILO-treated rats were imaged with a 2 T MRI scanner. T2HP and volume values were correlated with the number of cells, mossy fiber sprouting, and spontaneous recurrent seizures (SRS) frequency over the 9 months following status epilepticus (SE). Compared to controls, KA-treated rats had unaltered T2HP, pronounced reduction in hippocampal volume and concomitant cell reduction in granule cell layer, CA1 and CA3 at 3 months post SE. In contrast, hippocampal volume was unchanged in PILO-treated animals despite detectable increased T2HP and cell loss in granule cell layer, CA1 and CA3. In the following 6 months, MRI hippocampal volume remained stable with increase of T2HP signal in the KA-treated group. The number of CA1 and CA3 cells was smaller than age-matched CTL group. In contrast, PILO group had MRI volumetric reduction accompanied by reduction in the number of CA1 and CA3 cells. In this group, T2HP signal was unaltered at 6 or 9 months after status. Reductions in the number of cells were not progressive in both models. Notably, the SRS frequency was higher in PILO than in the KA model. The volumetry data correlated well with tissue damage in the epileptic brain, suggesting that MRI may be useful for tracking longitudinal hippocampal changes, allowing the assessment of individual variability and disease progression. Our results indicate that the temporal changes in hippocampal morphology are distinct for both models of TLE and that these are not significantly correlated to the frequency of SRS.

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy

Purpose:  Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese‐enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well‐established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE).

Manganese-Enhanced MRI: Biological Applications in Neuroscience

Magnetic resonance imaging (MRI) is an excellent non-invasive tool to investigate biological systems. The administration of the paramagnetic divalent ion manganese (Mn2+) enhances MRI contrast in vivo. Due to similarities between Mn2+ and calcium (Ca2+), the premise of manganese-enhanced MRI (MEMRI) is that the former may enter neurons and other excitable cells through voltage-gated Ca2+ channels. As such, MEMRI has been used to trace neuronal pathways, define morphological boundaries, and study connectivity in morphological and functional imaging studies. In this article, we provide a brief overview of MEMRI and discuss recently published data to illustrate the usefulness of this method, particularly in animal models.

Sex-related long-term behavioral and hippocampal cellular alterations after nociceptive stimulation throughout postnatal development in rats

Early noxious stimuli may alter the neurogenesis rate in the dentate gyrus and the behavioral repertoire of adult rats. This study evaluated the long-term effects of noxious stimulation, imposed in different phases of development, on nociceptive and anxiety-like behaviors, hippocampal activation, cell proliferation, hippocampal BDNF and plasma corticosterone levels in 40 day-old male and female adolescents. Noxious stimulation was induced by intra-plantar injection of Complete Freunds adjuvant (CFA), on postnatal days (P) 1 (group P1), 8 (P8) or 21 (P21). Control animals were not stimulated in any way. On P21 a subset of animals from each group received BrdU and was perfused on P40 for identification of proliferating cells in the granule cell layer of the dentate gyrus. Another subset of rats was subjected to behavioral testing on P40 and one week later, to magnetic resonance imaging (MRI) acquisition. Noxious stimulation evoked hypoalgesia in adolescents, mainly in females (P < 0.02), reflected by greater latency to withdraw the paw and less paw lickings in the hot plate test than controls (P < 0.001). It also resulted in more time spent in the open arms, e.g., less anxiety-like behavior than controls (P < 0.01), especially in females (P < 0.01, compared with males). Proliferative cell rate in the dentate gyrus was the highest in P8 males and females (P < 0.001), with males exhibiting more proliferation than females on P1 and P8, which was directly related to the hippocampal levels of BDNF and inversely related to plasma corticosterone. Sex differences were also detected in manganese-enhanced MRI signal, which was more prominent in P1 females than males (P < 0.01). This study represents the first step of investigation on the cellular basis of the sex-dependent long-term consequences of nociceptive stimuli in newborns.

In vivo magnetic resonance imaging tracking of C6 glioma cells labeled with superparamagnetic iron oxide nanoparticles

OBJECTIVE The aim of the current study was to monitor the migration of superparamagnetic iron oxide nanoparticle (SPION)-labeled C6 cells, which were used to induce glioblastoma tumor growth in an animal model, over time using magnetic resonance imaging (MRI), with the goal of aiding in tumor prognosis and therapy. METHODS Two groups of male Wistar rats were used for the tumor induction model. In the first group (n=3), the tumors were induced via the injection of SPION-labeled C6 cells. In the second group (n=3), the tumors were induced via the injection of unlabeled C6 cells. Prussian Blue staining was performed to analyze the SPION distribution within the C6 cells in vitro. Tumor-inducing C6 cells were injected into the right frontal cortex, and subsequent tumor monitoring and SPION detection were performed using T2- and T2*-weighted MRI at a 2T field strength. In addition, cancerous tissue was histologically analyzed after performing the MRI studies. RESULTS The in vitro qualitative evaluation demonstrated adequate distribution and satisfactory cell labeling of the SPIONs. At 14 or 21 days after C6 injection, a SPION-induced T2- and T2*-weighted MRI signal reduction was observed within the lesion located in the left frontal lobe on parasagittal topography. Moreover, histological staining of the tumor tissue with Prussian Blue revealed a broad distribution of SPIONs within the C6 cells. CONCLUSION MRI analyses exhibit potential for monitoring the tumor growth of C6 cells efficiently labeled with SPIONs.

Impact of neonatal anoxia on adult rat hippocampal volume, neurogenesis and behavior

Neonates that suffer oxygen deprivation during birth can have long lasting cognitive deficits, such as memory and learning impairments. Hippocampus, one of the main structures that participate in memory and learning processes, is a plastic and dynamic structure that conserves during life span the property of generating new cells which can become neurons, the so-called neurogenesis. The present study investigated whether a model of rat neonatal anoxia, that causes only respiratory distress, is able to alter the hippocampal volume, the neurogenesis rate and has functional implications in adult life. MRI analysis revealed significant hippocampal volume decrease in adult rats who had experienced neonatal anoxia compared to control animals for rostral, caudal and total hippocampus. In addition, these animals also had 55.7% decrease of double-labelled cells to BrdU and NeuN, reflecting a decrease in neurogenesis rate. Finally, behavioral analysis indicated that neonatal anoxia resulted in disruption of spatial working memory, similar to human condition, accompanied by an anxiogenic effect. The observed behavioral alterations caused by oxygen deprivation at birth might represent an outcome of the decreased hippocampal neurogenesis and volume, evidenced by immunohistochemistry and MRI analysis. Therefore, based on current findings we propose this model as suitable to explore new therapeutic approaches.

Reduced hippocampal manganese-enhanced MRI (MEMRI) signal during pilocarpine-induced status epilepticus: Edema or apoptosis?

Manganese-enhanced MRI (MEMRI) has been considered a surrogate marker of Ca(+2) influx into activated cells and tracer of neuronal active circuits. However, the induction of status epilepticus (SE) by kainic acid does not result in hippocampal MEMRI hypersignal, in spite of its high cell activity. Similarly, short durations of status (5 or 15min) induced by pilocarpine did not alter the hippocampal MEMRI, while 30 min of SE even reduced MEMRI signal Thus, this study was designed to investigate possible explanations for the absence or decrease of MEMRI signal after short periods of SE. We analyzed hippocampal caspase-3 activation (to evaluate apoptosis), T2 relaxometry (tissue water content) and aquaporin 4 expression (water-channel protein) of rats subjected to short periods of pilocarpine-induced SE. For the time periods studied here, apoptotic cell death did not contribute to the decrease of the hippocampal MEMRI signal. However, T2 relaxation was higher in the group of animals subjected to 30min of SE than in the other SE or control groups. This result is consistent with higher AQP-4 expression during the same time period. Based on apoptosis and tissue water content analysis, the low hippocampal MEMRI signal 30min after SE can potentially be attributed to local edema rather than to cell death.

Dmdmdx/Largemyd: a new mouse model of neuromuscular diseases useful for studying physiopathological mechanisms and testing therapies

SUMMARY Although muscular dystrophies are among the most common human genetic disorders, there are few treatment options available. Animal models have become increasingly important for testing new therapies prior to entering human clinical trials. The Dmdmdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD), presenting the same molecular and protein defect as seen in humans with the disease. However, this mouse is not useful for clinical trials because of its very mild phenotype. The mouse model for congenital myodystrophy type 1D, Largemyd, harbors a mutation in the glycosyltransferase Large gene and displays a severe phenotype. To help elucidate the role of the proteins dystrophin and LARGE in the organization of the dystrophin-glycoprotein complex in muscle sarcolemma, we generated double-mutant mice for the dystrophin and LARGE proteins. The new Dmdmdx/Largemyd mouse model is viable and shows a severe phenotype that is associated with the lack of dystrophin in muscle. We tested the usefulness of our new mouse model for cell therapy by systemically injecting them with normal murine mesenchymal adipose stem cells (mASCs). We verified that the mASCs were hosted in the dystrophic muscle. The new mouse model has proven to be very useful for the study of several other therapies, because injected cells can be screened both through DNA and protein analysis. Study of its substantial muscle weakness will also be very informative in the evaluation of functional benefits of these therapies.

Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx, Largemyd and mdx/Largemyd

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.

Tumor growth analysis by magnetic resonance imaging of the C6 glioblastoma model with prospects for the assessment of magnetohyperthermia therapy

OBJECTIVE The objective was to establish a pattern of tumor growth of the C6 model of glioblastoma multiform in Wistar rats via magnetic resonance imaging (MRI) for the subsequent verification of tumor volume reduction due to magnetic hyperthermia therapy. METHODS Young male Wistar rats weighing between 250 and 300 g were used for the C6 model. After the rats were anesthetized (55 mg/ kg ketamine and 11 mg/kg xylazine), C6 lineage tumorigenic cells suspended in culture medium (10(5) cells in 10 microl) were stereotaxically injected into the right frontal cortex (bregma coordinates: 2.0 mm anteroposterior, 3.0 mm laterolateral, and 2.5 mm depth) of the rats using a Hamilton syringe. For the control group, the rats were injected with culture medium without cells. MRI scans were performed at 14, 21, and 28 d after the injection using a 2.0 T MRI scanner (Bruker BioSpec, Germany). The animals were anesthetized with 55 mg/kg ketamine and 11 mg/kg xylazine before being examined. Coronal multilayers were acquired using a standard spin echo sequence with the following parameters: repetition/echo time = 4.000 ms/67.1 ms, field of view = 3.50, matrix = 192, slice thickness = 0.4 mm, and slice separation = 0 mm. RESULTS The MRI analysis enabled a clear visualization of the tumor mass, and it was possible to establish the tumor volume parameters on the various days that were examined. The volume at 14 d after induction was 13.7 +/- 2.5 mm3. On days 21 and 28, the tumor volumes were 31.7 +/- 6.5 mm3 and 122.1 +/- 11.8 mm3, respectively. CONCLUSION These results demonstrated that it is possible to evaluate the C6 model tumor volume in rats, which will allow for the future implementation and verification of magnetic hyperthermia therapy.