Targeted UHPLC-ESI-MS/MS Analysis of Selected Neurotransmitters, Tryptophan and Its Metabolite Kynurenine in Tau Transgenic Rat Brain Tissue: A Pivotal Study

Neurotransmitters (NT) are widely distributed in the central nervous system. These molecules are important for many physiological processes and the function of the immune system. Imbalance of NT are linked to numerous neurological disorders and diseases, including tauopathies. Here, a targeted approach based on on-line combination of ultra-high performance liquid chromatography with tandem mass spectrometry was validated and applied to the quantitative analysis of nine NT (acetylcholine, choline, aspartic acid, asparagine, glutamic acid, glutamine, pyroglutamate, γ-aminobutyric acid, N-acetyl-L-aspartic acid), tryptophan and its metabolite kynurenine in brain tissue samples of a rat model for tauopathy. The applied analytical method was characterized by excellent validation parameters for all analytes, such as limits of detection in the range of 0.01–1.70 µg/mL, regression coefficients of the calibration curves ≥ 0.9946, intra-day and inter-day precision expressed as coefficient of variation in the range of 0.6–11.9% and 0.6–14.4%, and accuracy in the range of 87.6–107.1% and 87.2–119.6%. Our analytical approach led to the identification of increased levels of choline and γ-aminobutyric acid in pons, and elevated concentration levels of pyroglutamate in medulla oblongata. These findings indicate that NT could play a valuable role in the study and clarification of neuroinflammation and neurodegenerative diseases.

Countering the classical renin–angiotensin system

It is well-established that Ang-(1-7) counteracts the effects of Ang II in the periphery, while stimulating vasopressin release and mimicking the activity of Ang II in the brain, through interactions with various receptors. The rapid metabolic inactivation of Ang-(1-7) has proven to be a limitation to therapeutic administration of the peptide. To circumvent this problem, Alves et al. (Clinical Science (2021) 135(18), https://doi.org/10.1042/CS20210599) developed a new transgenic rat model that overexpresses an Ang-(1-7)-producing fusion protein. In this commentary, we discuss potential concerns with this model while also highlighting advances that can ensue from this significant technical feat.

A Transgenic Rat for Noninvasive Assessment of Chondrogenesis in Vivo

Objective To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. Design A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. Results Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. Conclusions This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized—GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.

Enduring Effects of Conditional Brain Serotonin Knockdown, Followed by Recovery, on Adult Rat Neurogenesis and Behavior

Serotonin (5-hydroxytryptamine, 5-HT) is a crucial signal in the neurogenic niche of the hippocampus, where it is involved in antidepressant action. Here, we utilized a new transgenic rat model (TetO-shTPH2), where brain 5-HT levels can be acutely altered based on doxycycline (Dox)-inducible shRNA-expression. On/off stimulations of 5-HT concentrations might uniquely mirror the clinical course of major depression (e.g., relapse after discontinuation of antidepressants) in humans. Specifically, we measured 5-HT levels, and 5-HT metabolite 5-HIAA, in various brain areas following acute tryptophan hydroxylase 2 (Tph2) knockdown, and replenishment, and examined behavior and proliferation and survival of newly generated cells in the dentate gyrus. We found that decreased 5-HT levels in the prefrontal cortex and raphe nuclei, but not in the hippocampus of TetO-shTPH2 rats, lead to an enduring anxious phenotype. Surprisingly, the reduction in 5-HT synthesis is associated with increased numbers of BrdU-labeled cells in the dentate gyrus. At 3 weeks of Tph2 replenishment, 5-HT levels return to baseline and survival of newly generated cells is unaffected. We speculate that the acutely induced decrease in 5-HT concentrations and increased neurogenesis might represent a compensatory mechanism.

Reduced motivation for social contact in Disrupted-in-schizophrenia transgenic rats

The Disrupted-in-schizophrenia 1 (DISC1) signaling pathway is considered to play a key role in schizophrenia, depression, autism and other psychiatric disorders. DISC1 is involved in regulating the dopaminergic neurotransmission in, among others, the mesolimbic reward system. A transgenic rat line tgDISC1 has been introduced as a model system to study behavioral phenotypes associated with abnormal DISC1 pathways. Here, we evaluated the impact of impaired DISC1 signaling on social (social interaction) and non-social (sucrose) reward preferences in the tgDISC1 animal model. In a plus-maze setting, rats chose between the opportunity for social interaction with an unfamiliar juvenile conspecific (social reward) or drinking sweet solutions with variable sucrose concentrations (non-social reward). tgDISC1 rats differed from wild-type rats in their social, but not in their non-social reward preferences. Specifically, DISC1 rats showed a lower interest in interaction with the juvenile conspecific, but did not differ from wild-type rats in their preference for higher sucrose concentrations. These results suggest that disruptions of the DISC1 pathway that is associated with altered dopamine transmission in the brain result in selective deficits in social motivation seen in neuropsychiatric illness.

Traumatic Brain Injury Exacerbates Alzheimer's Disease Pathology in the Retinas of TgF344-AD Rats

Alzheimer's disease (AD) is a neurodegenerative condition that affects 6.2 million people age 65 and older in the U.S. alone, and is the leading cause of dementia. Moreover, AD can lead to visual impairment, and AD histopathology also manifests in the retina. However, the factors that modulate AD pathophysiology and lead to varied susceptibility and presentation in the population are not well understood. In this context, traumatic brain injury (TBI), which can arise from sport concussions, military combat, and other causes, is associated with a 2.3-fold higher risk of developing AD and AD-related dementias (ADRD). Thus, we set out to evaluate the effects of TBI, AD, and their combination, on retinal histopathology. Several animal models have been developed to investigate the mechanisms underlying AD, but many have been limited by imperfect recapitulation of human pathology, and no model of TBI-associated AD (AD-TBI) has been characterized. To address this gap, we generated an innovative model of AD-TBI by taking advantage of a transgenic rat model (Tg-F344-AD) shown to recapitulate the main features of human AD pathology, and combining it with a two-time unilateral controlled cortical impact paradigm to mimic repetitive mild TBI (rmTBI). Histopathological analyses at four months post-impact confirm the presence of AD markers in transgenic retinas, and an increased severity of AD pathology due to TBI. Together, these results contribute to our understanding of the effects of TBI on AD retinopathy, with implications for patient care and therapeutic development.

Stereological Changes in Microvascular Parameters in Hippocampus of a Transgenic Rat Model of Alzheimer’s Disease

Background: Microcirculatory factors play an important role in amyloid-β (Aβ)-related neuropathology in Alzheimer’s disease (AD). Transgenic (Tg) rat models of mutant Aβ deposition can enhance our understanding of this microvascular pathology. Objective: Here we report stereology-based quantification and comparisons (between- and within-group) of microvessel length and number and associated parameters in hippocampal subregions in Tg model of AD in Fischer 344 rats and non-Tg littermates. Methods: Systematic-random samples of tissue sections were processed and laminin immunostained to visualize microvessels through the entire hippocampus in Tg and non-Tg rats. A computer-assisted stereology system was used to quantify microvessel parameters including total number, total length, and associated densities in dentate gyrus (DG) and cornu ammonis (CA) subregions. Results: Thin hair-like capillaries are common near Aβ plaques in hippocampal subregions of Tg rats. There are a 53% significant increase in average length per capillary across entire hippocampus (p≤0.04) in Tg compared to non-Tg rats; 49% reduction in capillary length in DG (p≤0.02); and, higher microvessel density in principal cell layers (p≤0.03). Furthermore, within-group comparisons confirm Tg but not non-Tg rats have significant increase in number density (p≤0.01) and potential diffusion distance (p≤0.04) of microvessels in principal cell layers of hippocampal subregions. Conclusion: We show the Tg deposition of human Aβ mutations in rats disrupts the wild-type microanatomy of hippocampal microvessels. Stereology-based microvascular parameters could promote the development of novel strategies for protection and the therapeutic management of AD.

Hemodynamic phenotyping of transgenic rats with ubiquitous expression of an angiotensin-(1-7)-producing fusion protein

Activation of the angiotensin (Ang) converting enzyme 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay were similar to control SD rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure, assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma ANP and higher urinary sodium concentration. Evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decreased total peripheral resistance. TG7371 rats also presented increased cardiac and global sympathetic tone, increased plasma AVP levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower BP. Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.