Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans

Central Nervous Activity upon Systemic Salicylate Application in Animals with Kanamycin-Induced Hearing Loss - A Manganese-Enhanced MRI (MEMRI) Study

This study investigated the effect of systemic salicylate on central auditory and non-auditory structures in mice. Since cochlear hair cells are known to be one major target of salicylate, cochlear effects were reduced by using kanamycin to remove or impair hair cells. Neuronal brain activity was measured using the non-invasive manganese-enhanced magnetic resonance imaging technique. For all brain structures investigated, calcium-related neuronal activity was increased following systemic application of a sodium salicylate solution: probably due to neuronal hyperactivity. In addition, it was shown that the central effect of salicylate was not limited to the auditory system. A general alteration of calcium-related activity was indicated by an increase in manganese accumulation in the preoptic area of the anterior hypothalamus, as well as in the amygdala. The present data suggest that salicylate-induced activity changes in the auditory system differ from those shown in studies of noise trauma. Since salicylate action is reversible, central pharmacological effects of salicylate compared to those of (permanent) noise-induced hearing impairment and tinnitus might induce different pathophysiologies. These should therefore, be treated as different causes with the same symptoms

Activity Dependent Changes In Functional And Morphological Characteristics Among Presympathetic Neurons Of The Rostral Ventrolateral Medulla

A sedentary lifestyle is a major risk factor for the development of cardiovascular disease (CVD), the leading cause of death among Americans. Increasing evidence implicates increased sympathetic nerve activity (SNA) as the link between a sedentary lifestyle and CVD. The research presented in this dissertation examines the region of the brainstem known as the rostral ventrolateral medulla (RVLM) and how its regulation of SNA changes as a result of sedentary conditions. Our group has previously reported that sedentary conditions enhance splanchnic SNA in response to pharmacologically induced decreases in blood pressure or by direct activation of the RVLM via microinjection of the amino acid glutamate. More recently, our group has published the first evidence of overt structural differences in phenotypically identified RVLM neurons from sedentary versus physically active rats. Although collectively these studies suggest that a sedentary lifestyle results in increased activity and sensitivity of presympathetic RVLM neurons involved in blood pressure regulation, direct evidence of this proposed mechanism for the observed increased splanchnic SNA is lacking. The studies presented in this dissertation use in vivo characterization and juxtacellular labeling of RVLM neurons to examine the potential mechanistic connection and physiological relevance of overt changes in their structure and function and how they relate to enhanced SNA in sedentary versus physically active rats. These cross sectional studies are complemented by longitudinally based studies of in vivo neuronal activity in the RVLM utilizing manganese-enhanced magnetic resonance imaging (MEMRI). The information gained from these studies will contribute to our understanding of how a sedentary lifestyle contributes to the development of CVD and may provide information on new therapeutic targets in the brain to prevent or slow the progression of CVD

Volumetric Manganese Enhanced Magnetic Resonance Imaging in mice (mus musculus)

The present doctoral thesis introduces a method for semi-automatic volumetric analysis of the hippocampus and other distinct brain regions in laboratory mice. The method of volumetric manganese enhanced magnetic resonance imaging (vMEMRI) makes use of the paramagnetic property of the manganese ion, Mn2+, which results in a positive contrast enhancement of specific brain areas on the MR image and enables a more detailed image of brain morphology. The chemical similarity of Mn2+ to Calcium leads to an accumulation of Mn2+ in excited cells and consequentially an enhanced signal in certain brain regions in an activity dependent manner. However, one major drawback for vMEMRI is the toxicity of Mn2+. Therefore, the aims of the thesis have been: (1) Establishment of a MEMRI protocol in mice (2) Optimization of a Mn2+ application procedure to reduce toxic side effects (3) Development of an automatized method to determine hippocampal volume (4) Validation of vMEMRI analysis (5) Application of volumetric analysis in mouse models of psychopathology This thesis splits into 3 studies. Study 1 deals with Mn2+ toxicity and introduces an application method that considerably reduces the toxic side effects of Mn2+. Study 2 validates vMEMRI as a method to reliably determine hippocampal volume and explores its utilization it in animals with genetically and chemically modified hippocampi. Study 3 displays the application vMEMRI in a mouse model of a psychiatric disorder. Study 1 shows that a single application of Mn2+ in dosages used in current MEMRI studies leads to considerable toxic side effects measurable with physiological, behavioral and endocrine markers. In contrast, a fractionated application of a low dose of Mn2+ is proposed as an alternative to a single injection of a high dose. Repeated application of low dosages of 30 mg/kg Mn2+ showed less toxic side effects compared to the application schemes with higher dosages of 60 mg/kg. Additionally, the best vMEMRI signal contrast was seen for an injection protocol of 30 mg/kg 8 times with an inter-injection interval of 24 h (8x30/24 protocol). The impact of the 8x30/24 application protocol on longitudinal studies was tested by determining whether learning processes are disturbed. Mice were injected with the 8x30/24 protocol 2 weeks prior to receiving a single footshock. Manganese injected mice showed less contextual freezing to the shock context and a shock context reminder one month after shock application. Furthermore, mice showed increased hyperarousal and no avoidance of shock context related odors. This impairment in fear conditioning indicates a disturbed associative learning of Mn2+ injected mice. Therefore, it was investigated whether Mn2+ application shows a specific disturbance of hippocampus dependent learning. Mice were subjected to habitual and spatial learning protocols 12 h after each injection in a water cross-maze. There was no impairment in learning protocols which allowed for hippocampus-independent habitual learning. However, Mn2+ injected mice were specifically impaired in the hippocampus-dependent spatial learning protocol. Furthermore, it was shown that only mice with higher Mn2+ accumulation showed this impairment. Altogether, the results of this chapter argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects. However, the treatment procedure has to be further improved to allow for an analysis of hippocampus-dependent learning processes as well. Because of the potential side effects, the vMEMRI method was applied as a final experiment in study 2 and 3. Study 2 introduces the method of vMEMRI, which allows, for the first time, an in vivo semi-automatic detection of hippocampal volume. Hippocampal volume of mice with genetically altered adult neurogenesis and those with chemically lesioned hippocampi could be analyzed with vMEMRI. Even the highly variable differences in hippocampal volume of these animals could be detected with vMEMRI. vMEMRI data correlated with manually obtained volumes and are in agreement with previously reported histological findings, indicating the high reliability of this method. Study 3 investigates the ability of vMEMRI to detect even small differences in brain morphology by examining volumetric changes of the hippocampus and other brain structures in a mouse model of PTSD supplemented with enriched housing conditions. It was shown, that exposure to a brief inescapable foot shock led to a volume reduction in both the left hippocampus and right central amygdala two months later. Enriched housing decreased the intensity of trauma-associated contextual fear independently of whether it was provided before or after the shock. vMEMRI analysis revealed that enriched housing led to an increase in whole brain volume, including the lateral ventricles and the hippocampus. Furthermore, the enhancement of hippocampal volume through enriched housing was accompanied by the amelioration of trauma-associated PTSD-like symptoms. Hippocampal volume gain and loss was mirrored by ex vivo ultramicroscopic measurements of the hippocampus. Together, these data demonstrate that vMEMRI is able to detect small changes in hippocampal and central amygdalar volumes induced by a traumatic experience in mice. In conclusion, vMEMRI proves to be very reliable and able to detect small volumetric differences in various brain regions in living mice. vMEMRI opens up a great number possibilities for future research determining neuroanatomical structure, volumes and activity in vivo as well as the ability to repeatedly determine such characteristics within each subject, given an improvement of the Mn2+ treatment protocols to minimize potential toxic side effects

Synaptic uncoupling in the mouse cochlea induced by noise or cisplatin and its clinical relevance

The cochlea has an autonomous circadian clock that controls auditory function and increases vulnerability to noise trauma during the active phase. The mechanisms regulating the circadian control of the auditory system are poorly understood. Furthermore, it remains unclear whether the circadian vulnerability to noise also applies to other auditory insults such as cisplatin, an anticancer drug with strong ototoxic side-effects. Here, we investigated the interplay between glucocorticoids or glutamate homeostasis and noise- or cisplatin-induced hearing loss in the context of circadian influences. Circulating glucocorticoids are important synchronizers of clock rhythms and play a role in noise vulnerability. Our results showed that the removal of adrenal glands, and the subsequent suppression of circadian glucocorticoid rhythms, rescued the increased sensitivity to noise exposure at nighttime. The greater vulnerability to night-noise trauma was associated with a glucocorticoid-dependent regulation of inflammatory genes in the cochlea. Dexamethasone, a synthetic glucocorticoid receptor agonist, protected from noise damage when administered at daytime, coinciding with endogenous circulating glucocorticoid levels being low, but was not equally effective at nighttime, highlighting the relevance of considering the time of the day for assessing the effectiveness of a drug. In parallel, we hypothesized that disruption of the auditory glutamatergic synapse could exacerbate the circadian impact of auditory insults. In a first step, we characterized the synapse status in mice lacking the glutamate transporter GLAST and found that GLAST deficient mice have a pre-existing auditory synaptopathy, which led to an increased sound-evoked activity in the inferior colliculus, something that was not seen in wild-type mice. GLAST deficient mice also showed greater vulnerability to cisplatin-induced ototoxicity at nighttime, with a near complete loss of synaptic coupling to the inner hair cell. This effect could be due to the direct impact of cisplatin on the clock machinery since the administration of cisplatin on ex-vivo cochleae at nighttime altered the rhythms of the clock protein Period2 in a dose dependent manner. A meta-analysis identified five genes that are associated with cisplatin-mediated ototoxicity in cancer patients. Four of these were found circadian in the mouse cochlea, also suggesting their involvement in the circadian vulnerability to cisplatin. Overall, considering chronopharmacological approaches to cisplatin treatment could diminish the adverse side-effects on the auditory system and improve the life quality of cancer survivors

Alkoholin palkitseviin ominaisuuksiin ja amfetamiinin myrkkyvaikutuksiin liittyvien hermostollisten järjestelmien funktionaalinen kuvantaminen

Alcohol addiction is one of the most prevalent brain disorders in the world. A major hurdle for reducing alcohol-related harms and developing effective treatments is the poor understanding of neural processes responsible for the development of dependence and addiction. Alcohol has been shown to affect various neurotransmitter systems; however, the mesolimbic dopamine (DA) system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has been thought to play a key role in producing the reinforcing effects of alcohol. The VTA region has also been suggested to be the anatomical site for the interaction of the dopaminergic system with the opioidergic and γ-aminobutyric acid (GABA) systems. Here, manganese-enhanced magnetic resonance imaging (MEMRI) and behavioral tests were used to study drug-induced alterations in brain activity of the alcohol-preferring AA (Alko Alcohol) and heterogeneous Wistar rats. MEMRI is based on the ability of paramagnetic Mn2+ ions to accumulate in excitable neurons, thus enhancing the T1-weighted signal in activated brain regions. Mn2+ ions can also be transported anterogradely and retrogradely in neurons, released to the synaptic cleft, and taken up by other neurons. These properties allow MEMRI to measure long-term changes in brain activity, as well as map neural pathways involved in acute and long-term drug actions, including drug reward and toxicity. The AA rats exposed to alcohol compared to water controls displayed a widespread and persistent activation in brain regions that have been previously linked with alcohol reinforcement. Similarly, activity in neural pathways originating in the NAc and projecting caudally to the midbrain was enhanced in alcohol drinking rats. Moreover, this alcohol-induced activation was blocked by systemic naltrexone (NLX) administration. Comparison of naïve AA and Wistar rats revealed a lowered basal activity in the caudal linear nucleus (CLi) of AA rats, which was restored by voluntary alcohol drinking. The intra-CLi γ-aminobutyric acid type A receptor (GABAA) agonist muscimol produced a dose-dependent increase in alcohol drinking, blocked by co-administration of the GABAA antagonist bicuculline, suggesting that the CLi GABAergic system is involved in the regulation of alcohol reward. MEMRI was also employed for assessing stimulant-induced toxicity. Methamphetamine and mephedron displayed disparate effects on brain activity, as methamphetamine produced widespread decreases in activity, whereas mephedron increased activity in limited brain areas. Taken together, the use of MEMRI for mapping alcohol- and stimulant-induced alterations in functional brain activity revealed networks and specific pathways that have potential for guiding further translational efforts to develop medications for drug abuse disorders, as well as for evaluating drug-induced toxicity.Alkoholismi on maailman yleisimpiä aivosairauksia. Alkoholin aiheuttamien haittojen vähentämistä ja tehokkaiden hoitomuotojen kehittämistä haittaa se, että päihderiippuvuuden kehittymiseen vaikuttavat aivojen toiminnan muutokset ymmärretään yhä huonosti. Alkoholin vaikutukset syntyvät monien hermoston välittäjäaineiden toiminnan kautta, ja etenkin nk. mesolimbisellä dopamiinijärjestelmällä on arveltu olevan keskeinen rooli alkoholin tuottamassa mielihyvässä. Töissämme käytettiin mangaanitehosteista magneettiresonanssikuvantamista (MRI) selvitettäessä alkoholin ja stimulanttien vaikutuksia alkoholiin mieltyneiden AA-rottien ja normaalien Wistar-rottien aivoissa. Kyseinen kuvantamismenetelmä perustuu siihen, että magneettisia ominaisuuksia omaavat mangaani-ionit kulkeutuvat hermopäätteiden kalsiumkanavien kautta hermosoluihin niiden aktivoituessa. Mangaani-ioneja sisältävien hermosolujen muodostamat radat ja aivoalueet tuottavat mitattavissa olevan signaalin. Mangaani voi myös siirtyä synapsiraon ylitse viereiseen hermosoluun ja tuottaa siten MRI-kuvan aktiivisista hermoverkoista. Viikkoja kestänyt alkoholin juominen johti aivojen aktiivisuusmuutoksiin sellaisilla aivojen alueilla, joiden on aikaisemmin oletettu liittyvän alkoholin positiivisiin vaikutuksiin. Selvitettäessä tarkemmin yksittäisten hermoratojen merkitystä havaittiin, että etuaivojen accumbens-tumakkeen keskiaivoihin lähettävä rata aktivoitui alkoholin juomisen seurauksena, kun taas alkoholismin hoidossa käytettävä lääkeaine, naltreksoni, vähensi alkoholin aiheuttamaa aktivaatiota. Verrattaessa alkoholiin mieltyneitä rottia tavalliseen rottakantaan löydettiin keskiaivoista tumake (kaudaalinen lineaaritumake), jonka aktiivisuus oli ennen alkoholin juomista tavallista alhaisempi, mutta jota alkoholi aktivoi. Kun tähän tumakkeeseen annettiin ainetta, joka salpasi gamma-aminovoihapon (GABA) vastaanottokohdat, alkoholin kulutus lisääntyi huomattavasti. Tämä löydös viittasi keskiaivojen GABA-järjestelmän merkitykseen alkoholin kulutuksen säätelyssä. Kaikkiaan kehittämämme mangaanitehosteinen magneettiresonanssikuvantaminen tuotti uutta tietoa alkoholin juomista säätelevistä hermoradoista ja yksittäisistä aivojen alueista. Tätä tietoa voidaan käyttää hyväksi, kun suunnitellaan ja testataan alkoholismin hoitoon tarkoitettuja lääkeaineita

MULTIMODAL ASSESSMENT OF CETACEAN CENTRAL NERVOUS AUDITORY PATHWAYS WITH EMPHASIS ON FORENSIC DIAGNOSTICS OF ACOUSTIC TRAUMA

Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment.Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment

The effect of sodium salicylate on the level of GABAB receptor subunits in the rat\u27s central auditory system

Tinnitus is a phantom sensation of sound in the absence of acoustic stimulus and can be induced by ototoxic drugs such as sodium salicylate (SS). Tinnitus is likely related to hyperactivity in central auditory structures. In the central nervous system, activity is dependent on excitatory/inhibitory neurotransmission. Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter. It can activate the type-B GABAergic receptor (GABA B receptor), which can control the level of excitation/inhibition through regulating the release of neurotransmitters and lowering the cell membrane potential. I hypothesized that SS affects hearing by changing the level and distribution of the GABA B receptor in central auditory structures. My results from immunohistochemical and western blotting experiments revealed that SS reduced the level of GABA B receptors in all major auditory structures. The reduction was observed in both neuronal cell bodies regions and areas containing axonal and dendritic fibers of neurons