Brain injections of glial cytoplasmic inclusions induce a multiple system atrophy-like pathology.

Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson's disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development.

Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates.

In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson's disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson's disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson's disease.

Regeneration of lingual musculature in rats using myoblasts over porcine bladder acellular matrix.

OBJECTIVES: To use tissue engineering muscle repair (TEMR) for regenerating the lingual musculature of hemiglossectomized rats using neonatal myoblasts (NM) on porcine acellular urinary bladder matrix (AUBM). MATERIAL AND METHODS: The study used 80 male rats. A volumetric muscle loss (VML) injury was created on the left side of the tongue. The rats were randomized into four groups: Group 1 (AUBM + myoblasts); Group 2 (AUBM); Group 3 (myoblasts); and Group 4 (control). NM were obtained from neonatal rats. The animals were weighed on day 0 and just before euthanasia. Five rats in each group were euthanized at days 2, 14, 28, and 42; the tongues were prepared for morphometric analysis, postoperative left hemitongue weight, and immunohistochemical analysis (desmin, CD-31, and anti-neurofilament). RESULTS: The weight gain from greatest to least was as follows: AUBM + myoblasts > myoblasts > AUBM > control. The tongue dorsum occupied by VML, and difference in mg between control side and intervened side from least to great was as follows: AUBM + myoblasts < myoblasts < AUBM < control. The order from highest to lowest antibody positivity was as follows: AUBM + myoblasts > myoblasts > AUBM > control. CONCLUSION: The use of porcine AUBM and NM for the regeneration of lingual musculature was found to be an effective TEMR treatment for repairing tongue VML injury.

Ultrasonography study of the skin wound healing process in gilthead seabream (Sparus aurata).

This work aimed to carry out an in vivo study of the skin healing process in gilthead seabream (Sparus aurata) after being experimentally wounded. Firstly, the structure of normal skin was studied by real-time ultrasonography (Vevo Lab, VisualSonics) and light microscopy. Besides this, experimental wounds were made on the left flank of each fish with a circular biopsy punch (8 mm diameter) below the lateral line. The healing process was assessed on live fish at 0, 6, 11 and 23 days post-wounding using the real-time ultrasonography in B-mode and Power Doppler mode (Vevo 3100 FUJIFILM, VisualSonics). Through the ultrasonography images, both the skin structure and the evolution of the changes that wounds originated in the surrounding tissues were studied in vivo over time. Concomitantly, the pattern of neovascularization in the wounded area was followed during the healing process and it was demonstrated that, although the neovascularization started very early after the skin damage, it was increased in wounded areas from day 11 post-wounding onwards. The results obtained proved the utility and power of using ultrasounds in fish to evaluate in vivo complex biological processes in real time, which are difficult to study by other methodologies. The present data shed some light on the reparation of external injuries in aquatic vertebrates.

The Use of Human Sterilized Crushed Tooth Particles Compared with BTCP Biomaterial and Empty Defects in Bone Formation inside Critical Rabbit Calvaria Sites.

This study aimed to assess the bone regeneration of critical-size defects in rabbit calvaria filled with freshly crushed extracted teeth, comparing them with BTCP biomaterial and empty sites. Materials and methods: Twenty-one female New Zealand rabbits were used in this study. Two critical-size defects 6 mm in size were created in the skull bone, each with a 3 mm separation between them. Three experimental groups were evaluated: Group A (human sterilized crushed teeth granules alone), Group B (Bioner Bone, Bioner Sitemas Implantológicos), and Group C (unfilled defects). The animals were sacrificed at 4 and 8 weeks. Evaluation of the samples involved histological and histomorphometric analyses with radiographic evaluation. The histological evaluation showed a higher volume reduction in Group A compared with Group B (p < 0.05) and Control. Group A showed the highest values for cortical closure and bone formation around the particles, followed by Group B and Group C (p < 0.05). Within the limitations of this animal study, we can conclude that the use of human tooth particles leads to increased bone formation and reduced connective tissue in critical-size defects in rabbit calvaria when compared to BTCP biomaterial. The calvarial model is a robust base for the evaluation of different biomaterials.

Cortical Lewy body injections induce long-distance pathogenic alterations in the non-human primate brain.

Aggregation of α-synuclein (α-syn) is the cornerstone of neurodegenerative diseases termed synucleinopathies, which include Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA). These synucleinopathies are characterized by the deposit of aggregated α-syn in intracellular inclusions observable in neurons and glial cells. In PD and DLB, these aggregates, predominantly located in neurons, are called Lewy Bodies (LBs). These LBs are one of the pathological hallmarks of PD and DLB, alongside dopaminergic neuron loss in the substantia nigra. Previous studies have demonstrated the ability of PD patient-derived LB fractions to induce nigrostriatal neurodegeneration and α-syn pathology when injected into the striatum or the enteric nervous system of non-human primates. Here, we report the pathological consequences of injecting these LB fractions into the cortex of non-human primates. To this end, we inoculated mesencephalic PD patient-derived LB fractions into the prefrontal cortex of baboon monkeys terminated one year later. Extensive analyses were performed to evaluate pathological markers known to be affected in LB pathologies. We first assessed the hypothesized presence of phosphorylated α-syn at S129 (pSyn) in the prefrontal cortices. Second, we quantified the neuronal, microglial, and astrocytic cell survival in the same cortices. Third, we characterized these cortical LB injections' putative impact on the integrity of the nigrostriatal system. Overall, we observed pSyn accumulation around the injection site in the dorsal prefrontal cortex, in connected cortical regions, and further towards the striatum, suggesting α-syn pathological propagation. The pathology was also accompanied by neuronal loss in these prefrontal cortical regions and the caudate nucleus, without, however, loss of nigral dopamine neurons. In conclusion, this pilot study provides novel data demonstrating the toxicity of patient-derived extracts, their potential to propagate from the cortex to the striatum in non-human primates, and a possible primate model of DLB.

Study of the Effect of Photobiomodulation on a Skin Repair Model in SKH-1 Mice.

Background and objective: Laser applied at low power (400-1100 nm), currently named photobiomodulation (PBM), is a noninvasive therapy to speed up wound healing. The purpose of this study was whether two different laser PBM delivery protocols would impact the skin wound healing in a mouse model. Materials and methods: A total of 24 SKH-1 mice were divided into three groups: Group 1 (control: untreated ulcers), Group 2 (a single postsurgical laser application), and Group 3 (laser each other day for 10 days; total five applications). Laser parameters were 940 nm, 0.4 W, 10 mm spot size, 0.008 J/cm2, 300 sec/wound. Each animal received two skin wounds which were photographed on days 0, 5, and 10 to determine wound closure (ImageJ). Half of the animals in each group were sacrificed on day 5 and the other half on day 10. Samples were routinely processed for histological analysis (re-epithelization, angiogenesis, granulation tissue formation, inflammation, and collagen deposition). Results: The closure of the wounds at the end of the experiment in the animals photobiostimulated each other day was more advanced than in the controls and in those treated only once, in both the macroscopic and microscopic studies. Angiogenesis was higher in both treated groups than in the control in the first study time (day 5). However, inflammation, maturation of the granulation tissue, and collagen deposition only improved when the laser was applied each other day. Conclusions: In our study, with the parameters used, PBM improved the healing of skin wounds when applied every other day and not in a single dose.

Assessment of hepatic function, perfusion and parenchyma attenuation with indocyanine green, ultrasound and computed tomography in a healthy rat model: Preliminary determination of baseline parameters in a healthy liver.

BACKGROUND: Defining reference intervals in experimental animal models plays a crucial role in pre-clinical studies. The hepatic parameters in healthy animals provide useful information about type and extension of hepatic damage. However, in the majority of the cases, to obtain them require an invasive techniques. Our study combines these determinations with dynamic functional test and imaging techniques to implement a non-invasive protocol for liver evaluation. The aim of the study was to determine reference intervals for hepatic function, perfusion and parenchyma attenuation with analytical and biochemical blood parameters, indocyanine green, ultrasound and computed tomography in six healthy SD rats. METHODS: Six males healthy SD rats were followed for 4 weeks. To determine hepatic function, perfusion and parenchyma attenuation analytical and biochemical blood parameters, indocyanine green, ultrasound and computed tomography were studied. Results were expressed as Means ± standard error of mean (SEM). The significance of differences was calculated by using student t-test, p < 0.05 was considered statistically significant. RESULTS: Indocyanine green clearance 5 and 10 minutes after its injection was 80.12% and 96.59%, respectively. Approximate rate of decay during the first 5 minutes after injection was 38% per minute. Hepatic perfusion evaluation with the high-frequency ultrasound was related to cardiovascular hemodynamic and renal perfusion. Portal area, hepatic artery resistance index, hepatic artery and portal peak systolic velocity and average between hepatic artery and porta was 3.41 ± 0.62 mm2, 0.57 ± 0.04 mm2/s, 693.24±102.53 mm2/s, 150.72 ± 17.80 mm2/s and 4.82 ± 0.96 mm2/s, respectively. Heart rate, cardiac output, left renal artery diammetre and renal blood flow were 331.01 ± 22.22 bpm, 75.58 ± 8.72 mL/min, 0.88 ± 0.04 mm2 and 13.65 ± 1.95 mm2/s. CT-scan hepatic average volume for each rat were 21.08±3.32, 17.57±2.76, 14.87±2.83 and 13.67±2.45 cm3 with an average attenuation coefficient of 113.51±18.08, 129,19±7.18, 141,47±1.95 y 151,67±1.2 HU. CONCLUSION: Indocyanine green and high-frequency ultrasound could be used in rats as a suitable marker of liver function. Computed tomography, through the study of raw data, help to characterize liver parenchyma, and could be a potential tool for early detection of liver parenchymal alterations and linear follow-up of patients. Further studies in rats with liver disease are necessary to verify the usefulness of these parameters.

Infrared-Emitting Multimodal Nanostructures for Controlled In Vivo Magnetic Hyperthermia.

Deliberate and local increase of the temperature within solid tumors represents an effective therapeutic approach. Thermal therapies embrace this concept leveraging the capability of some species to convert the absorbed energy into heat. To that end, magnetic hyperthermia (MHT) uses magnetic nanoparticles (MNPs) that can effectively dissipate the energy absorbed under alternating magnetic fields. However, MNPs fail to provide real-time thermal feedback with the risk of unwanted overheating and impeding on-the-fly adjustment of the therapeutic parameters. Localization of MNPs within a tissue in an accurate, rapid, and cost-effective way represents another challenge for increasing the efficacy of MHT. In this work, MNPs are combined with state-of-the-art infrared luminescent nanothermometers (LNTh; Ag2 S nanoparticles) in a nanocapsule that simultaneously overcomes these limitations. The novel optomagnetic nanocapsule acts as multimodal contrast agents for different imaging techniques (magnetic resonance, photoacoustic and near-infrared fluorescence imaging, optical and X-ray computed tomography). Most crucially, these nanocapsules provide accurate (0.2 °C resolution) and real-time subcutaneous thermal feedback during in vivo MHT, also enabling the attainment of thermal maps of the area of interest. These findings are a milestone on the road toward controlled magnetothermal therapies with minimal side effects.

Comparative Anatomy of the Nasal Cavity in the Common Dolphin Delphinus delphis L., Striped Dolphin Stenella coeruleoalba M. and Pilot Whale Globicephala melas T.: A Developmental Study.

Our goal was to analyze the main anatomical structures of the dolphin external nose and nasal cavity from fetal developmental stages to adult. Endoscopy was used to study the common development of the external nose and the melon, and nasal mucosa. Magnetic resonance imaging (MRI) and anatomical sections were correlated with anatomical sections. Computed tomography (CT) was used to generate 3D reconstructions of the nasal bones and nasal cavities to study its development. Dissections, histological and pathological studies were carried out on the nasal mucosa to understand its function. These results were compared with the horse. Endoscopy showed an external nose with two lips and the upper lip is divided by a groove due to the nasal septum and an obstruction of right nasal cavity was diagnosed in a newborn. Two diverticula (air sacs) were found in the nasal vestibule and an incisive recess (premaxillary sac) in the nasal cavity. These findings were corroborated by 3D reconstructions of the nasal cavities, MRI, anatomical sections and dissections. The presphenoid and ethmoid bones were fused at early stages of fetal development. The ethmoid is the last bone to ossify in the nasal cavity.

Radiological characterization of gilthead seabream (Sparus aurata) fat by X-ray micro-computed tomography.

In fish, the fat content contributes to promoting the nutritional and organoleptic characteristics of the flesh, which is crucial for consumer acceptance. Methods to predict the fat in fish are important in nutritional and physiological research, where body content is traditionally determined by dissection followed by chemical analysis. However, X-ray micro-computed tomography (micro-CT) provides three-dimensional information in a non-destructive way. This work aims to characterize radiologically the fat, in situ, in a widely cultivated marine species, gilthead seabream (Sparus aurata). To validate the method changes in fat content in a control group (fed) and another group (unfed for 60 days) were assessed. Fish images were acquired on an Albira SPECT/PET/CT preclinical-scanner. Image analysis and measurements were performed using the Carestream Molecular Imaging Albira CT system in conjunction with Pmod and Amide packages. By micro-CT analysis the density values were determined for the whole fish body (- 1,000 to + 2,500 HU, Hounsfield units), and density ranges for the fat in S. aurata were established from - 115 to + 50 HU. As expected, significant differences were found between fed and starved groups at 60 days. The present study confirms the usefulness of high-resolution morphological analysis for evaluating the presence and distribution of fat in this important fish species.

Evaluation of rat liver with ARFI elastography: In vivo and ex vivo study.

OBJECTIVE: The aim of this study was to compare in vivo vs ex vivo liver stiffness in rats with acoustic radiation force impulse (ARFI) elastography using the histological findings as the gold standard. METHODS: Eighteen male Wistar rats aged 16-18 months were divided into a control group (n = 6) and obese group (n = 12). Liver stiffness was measured with shear wave velocity (SWV) using the ARFI technique both in vivo and ex vivo. The degree of fibrosis, steatosis and liver inflammation was evaluated in the histological findings. Pearson's correlation coefficient was applied to relate the SWV values to the histological parameters. RESULTS: The SWV values acquired in the ex vivo study were significantly lower than those obtained in vivo (P < 0.004). A significantly higher correlation value between the degree of liver fibrosis and the ARFI elastography assessment was observed in the ex vivo study (r = 0.706, P < 0.002), than the in vivo study (r = 0.623, P < 0.05). CONCLUSION: Assessment of liver stiffness using ARFI elastography yielded a significant correlation between SWV and liver fibrosis in both the in vivo and ex vivo experiments. We consider that by minimising the influence of possible sources of artefact we could improve the accuracy of the measurements acquired with ARFI.

Demineralized Bone Matrix Coating Si-Ca-P Ceramic Does Not Improve the Osseointegration of the Scaffold.

The aim of this study was to manufacture and evaluate the effect of a biphasic calcium silicophosphate (CSP) scaffold ceramic, coated with a natural demineralized bone matrix (DBM), to evaluate the efficiency of this novel ceramic material in bone regeneration. The DBM-coated CSP ceramic was made by coating a CSP scaffold with gel DBM, produced by the partial sintering of different-sized porous granules. These scaffolds were used to reconstruct defects in rabbit tibiae, where CSP scaffolds acted as the control material. Micro-CT and histological analyses were performed to evaluate new bone formation at 1, 3, and 5 months post-surgery. The present research results showed a correlation among the data obtained by micro-CT and the histomorphological results, the gradual disintegration of the biomaterial, and the presence of free scaffold fragments dispersed inside the medullary cavity occupied by hematopoietic bone marrow over the 5-month study period. No difference was found between the DBM-coated and uncoated implants. The new bone tissue inside the implants increased with implantation time. Slightly less new bone formation was observed in the DBM-coated samples, but it was not statistically significant. Both the DBM-coated and the CSP scaffolds gave excellent bone tissue responses and good osteoconductivity.

Ultrasonography in Experimental Reproductive Investigations on Rats.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

Melanopsin-Containing or Non-Melanopsin-Containing Retinal Ganglion Cells Response to Acute Ocular Hypertension With or Without Brain-Derived Neurotrophic Factor Neuroprotection.

Purpose: To analyze the responses of different retinal ganglion cell (RGC) types to acute ocular hypertension (AOH) and intravitreal administration of brain-derived neurotrophic factor (BDNF). Methods: In adult albino rats, the anterior chamber of the left eye was cannulated with a needle connected to a saline container elevated 1½ meters above the eye for 75 minutes. Rats received 12 hours before a 5 µl intravitreal injection containing 5 µg BDNF in 1% albumin PBS or vehicle and were analyzed 3, 7, 14, or 45 days later. Both retinas were dissected as wholemounts and immunolabeled for melanopsin (to identify intrinsically photosensitive RGCs) or Brn3a (to identify all RGCs except melanopsin +RGCs). Results: During AOH there is ischemic damage and mechanical eye-globe deformation. Acute ocular hypertension results in a progressive loss of Brn3a+RGCs in the vehicle-treated retinas (39%, 35%, 25%, and 13% of the original value, at 3, 7, 14, or 45 days, respectively), whereas BDNF increases their survival to 81%, 73%, 59%, or 57% at the same time periods. In vehicle-treated retinas, 37% or 39% of m+RGCs survive at 14 or 45 days, respectively, whereas BDNF treatment increases their survival to 40% or 78% at the same time points. Conclusions: Different types of RGCs respond differently to AOH because Brn3a+RGCs die progressively, but m+RGCs do not. After a transient downregulation of melanopsin expression, their number remains constant and their survival is proportionally higher than that of Brn3a+RGCs. BDNF affords a permanent protection up to 45 days after AOH injury in both types of RGCs.

Radioprotective effects of lycopene and curcumin during local irradiation of parotid glands in Sprague Dawley rats.

Radiotherapy effectively treats cancers of the head and neck. We investigated the possible protective effects of lycopene and curcumin on the parotid glands of 40 female Sprague Dawley rats during irradiation. The study followed European Union regulations 86/609/EEC, 2010/63/EU for animal experimentation. The animals were divided into 4 groups: those treated with curcumin and radiation, those treated with lycopene and radiation, those treated with dimethyl sulphoxide (DMSO) and radiation, and those treated with radiation alone. All compounds were given intraperitoneally the day before irradiation. The total dose of radiation was 20Gy. Morphological and histopathological analyses showed less cell necrosis in the group treated with curcumin than in the other groups, but the difference was not significant. Analysis of structural damage to the parotid ducts and vacuolisation showed significant differences among all groups (p=0.023, p<0.01). Lycopene and curcumin given 24 hours before irradiation reduced the structural damage to the salivary glands. Further studies are needed to confirm these findings.