Animal Models Research Articles

Enhancement of enteric neural stem cell neurogenesis by glial cell-derived neurotrophic factor in experimental Hirschsprung's disease.

Stem cell therapy offers a promising solution for congenital diseases like Hirschsprung's disease (HSCR). Optimizing stem cell efficacy by modifying the cells and their environment is crucial, but in vitro culture conditions need to be further improved. Glial cell-derived neurotrophic factor (GDNF) plays an important role in neuronal survival, proliferation, migration and differentiation during enteric nervous system (ENS) development. In this study, the effects of GDNF on neurites derived from an Ednrb knockout model were investigated with the aim of enhancing the neurogenic potential of enteric neural crest cells (ENCCs). Neurospheres were generated form Ednrb+/+ (control) and Ednrb-/- mice at embryonic day13.5 (E13.5) with Sox10-green fluorescent protein (Venus) transgenic expression. These neurospheres were cultured in control media and neurospheres from Ednrb-/- were cultured with either control media or media supplemented with GDNF. ENCCs differentiation was assessed using immunofluorescence staining after 18days. GDNF-treated Ednrb-/- neurospheres showed increased size and higher density of Sox10-positive ENCCs compared to untreated Ednrb-/- neurospheres. GDNF also enhanced the distribution of both TUJ1-positive neurons and S100-positive glial cells. GDNF effectively enhanced the neurogenic potential of ENCCs from HSCR animal model. This finding is crucial for the development of cell therapy in HSCR.

Vitamin C supplementation improves placental function and alters placental gene expression in smokers

Maternal smoking during pregnancy (MSDP), driven by nicotine crossing the placenta, causes lifelong decreases in offspring pulmonary function and vitamin C supplementation during pregnancy prevents some of those changes. We have also shown in animal models of prenatal nicotine exposure that vitamin C supplementation during pregnancy improves placental function. In this study we examined whether vitamin C supplementation mitigates the effects of MSDP on placental structure, function, and gene expression in pregnant human smokers. Doppler ultrasound was performed in a subset of 55 pregnant smokers participating in the “Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function” (VCSIP) randomized clinical trial (NCT01723696) and in 33 pregnant nonsmokers. Doppler ultrasound measurements showed decreased umbilical vein Doppler velocity (Vmax) in placebo-treated smokers that was significantly improved in smokers randomized to vitamin C, restoring to levels comparable to nonsmokers. RNA-sequencing demonstrated that vitamin C supplementation to pregnant smokers was associated with changes in mRNA expression in genes highly relevant to vascular and cardiac development, suggesting a potential mechanism for vitamin C supplementation in pregnant smokers to improve some aspects of offspring health.

Lessons from IgA Nephropathy Models

IgA nephropathy (IgAN) is the most common type of primary glomerulonephritis worldwide; however, the underlying mechanisms of this disease are not fully understood. This review explores several animal models that provide insights into IgAN pathogenesis, emphasizing the roles of aberrant IgA1 glycosylation and immune complex formation. It discusses spontaneous, immunization, and transgenic models illustrating unique aspects of IgAN development and progression. The animal models, represented by the grouped ddY (gddY) mouse, have provided guidance concerning the multi-hit pathogenesis of IgAN. In this paradigm, genetic and environmental factors, including the dysregulation of the mucosal immune system, lead to increased levels of aberrantly glycosylated IgA, nephritogenic immune complex formation, and subsequent glomerular deposition, followed by mesangial cell activation and injury. Additionally, this review considers the implications of clinical trials targeting molecular pathways influenced by IgAN (e.g., a proliferation-inducing ligand [APRIL]). Collectively, these animal models have expanded the understanding of IgAN pathogenesis while facilitating the development of therapeutic strategies that are currently under clinical investigation. Animal-model-based studies have the potential to facilitate the development of targeted therapies with reduced side effects for IgAN patients.

Preclinical Testing Strategies for Epilepsy Therapy Development

The development of antiepileptogenic and disease-modifying treatments for epilepsy is a key goal of epilepsy research. Technological and scientific advances over the past two decades have seen the development of numerous therapeutic approaches, many of which show great promise in animal models. To facilitate and de-risk the translation of these promising approaches, however, rigorous preclinical testing is needed. For the present review, we discuss challenges and approaches to conduct preclinical testing of antiepileptogenic and disease-modifying treatments in animal models.

Sex as a biological variable in HIV-1 and schistosome co-infection.

The sex of people living with HIV-1 infection, schistosome infection, or both, is a fundamental determinant of their clinical outcomes and of how these two infections interact in the host. Data from longitudinal and cross-sectional human studies and animal models indicate that males with HIV-1 and schistosome co-infection excrete fewer schistosome eggs and might have higher HIV-1 RNA viral loads and greater liver damage. Females with schistosome infection appear to have higher risk of HIV-1 acquisition than females without, particularly in Schistosoma haematobium infection, and a greater risk of death in HIV-1 and schistosome co-infection. Greater transmission of HIV-1 to partners has been shown in both sexes in those with schistosome infection. Biological sex is a consequential factor affecting pathophysiological and clinical responses in HIV-1 and schistosome co-infection. Designing future analyses to incorporate sex is vital to optimise research and care for people living with HIV-1, schistosomes, and HIV-1 and schistosome co-infection.

Cerebral hyperactivation across the Alzheimer’s disease pathological cascade

Abstract Neuronal dysfunction in specific brain regions or across distributed brain networks is a known feature of Alzheimer’s disease. An often reported finding in the early stage of the disease is the presence of increased functional MRI (fMRI) BOLD signal under task conditions relative to cognitively normal controls, a phenomenon known as “hyperactivation”. However, research in the past decades yielded complex, sometimes conflicting results. The magnitude and topology of fMRI hyperactivation patterns have been found to vary across the preclinical and clinical spectrum of Alzheimer’s disease, including concomitant “hypoactivation” in some cases. These incongruences are likely due to a range of factors, including the disease stage at which the cohort is examined, the brain areas or networks studied, and the fMRI paradigm utilised to evoke these functional abnormalities. Additionally, a perennial question pertains to the nature of hyperactivation in the context of Alzheimer’s disease. Some propose it reflects compensatory mechanisms to sustain cognitive performance while others suggest it is linked to the pathological disruption of a highly regulated homeostatic cycle that contributes to, or even drives, disease progression. Providing a coherent narrative for these empirical and conceptual discrepancies is paramount to develop disease models, understand the synergy between hyperactivation and the Alzheimer’s disease pathological cascade, and tailor effective interventions. We first provide a comprehensive overview of functional brain changes spanning the course from normal ageing to the clinical spectrum of Alzheimer’s disease. We then highlight evidence supporting a close relationship between fMRI hyperactivation and in vivo markers of Alzheimer’s pathology. We primarily focus on task-based fMRI studies in humans, but also consider studies using different functional imaging techniques and animal models. We then discuss the potential mechanisms underlying hyperactivation in the context of Alzheimer’s disease, and provide a testable framework bridging hyperactivation, ageing, cognition, and the Alzheimer’s disease pathological cascade. We conclude with a discussion of future challenges and opportunities to advance our understanding of the fundamental disease mechanisms of Alzheimer’s disease, and the promising development of therapeutic interventions incorporating or aimed at hyperactivation and large-scale functional systems.

Cytochalasin H enhances sensitivity to gefitinib in non-small-cell lung cancer cells through inhibiting EGFR activation and PD-L1 expression

In our previous study, we have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove plant and found that CyH inhibited the proliferation of non-small cell lung cancer (NSCLC) cells. Recently, epidermal growth factor receptor (EGFR) activation and programmed cell death 1 ligand (PD-L1) expression have been demonstrated to mediate NSCLC resistance to gefitinib, first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI). Here, we further investigated the effect of CyH on EGFR activation, PD-L1 expression, and gefitinib sensitivity in NSCLC cell lines, A549 (wild-type EGFR), HCC827 (EGFR mutation), and NCI-H1975 (dual EGFR mutations and acquired gefitinib resistance) and animal model. Our results showed that CyH significantly inhibited EGFR activation and PD-L1 expression in NSCLC cells. Additionally, CyH dramatically promoted the inhibitory effect of gefitinib on the proliferation of A549 and HCC827 cells, and enhanced the sensitivity to gefitinib in NCI-H1975 cells. Moreover, CyH increased the inhibitory effect of gefitinib on EGFR activation and PD-L1 expression in HCC827 and NCI-H1975 cells. Animal experiments further demonstrated that CyH significantly promoted the inhibitory effect of gefitinib on the growth of NSCLC and the expression of Ki-67, p-EGFR, and PD-L1 in NCI-H1975 NSCLC xenograft tumors of nude mice. Furthermore, CyH inhibited the activation of JAK3/STAT signaling pathway. Taken together, our findings suggest that CyH promotes the sensitivity to gefitinib in NSCLC cells through the inhibition of EGFR activation and PD-L1 expression.

Cerebral haemodynamics and intracranial pressure during haemorrhagic shock and resuscitation with total endovascular balloon occlusion of the aorta in an animal model.

To assess changes of cerebral haemodynamic and intracranial pressure (ICP) in animals, with or without elevated ICP, during controlled haemorrhagic shock and resuscitation with Total REBOA (tREBOA). In 22 anaesthetized and normoventilated pigs, after placement of catheters for monitoring invasive proximal blood pressure (pMAP), ICP, and vital parameters, and 60min stabilisation phase, a controlled haemorrhagic shock (HS), was conducted. In 11 pigs (EICPG), an elevated ICP of 25-30 mmHg at the end HS was achieved by simulating an epidural mass. In 11 pigs (NICPG), the ICP was normal. tREBOA was then applied for 120min. The changes of pMAP and ICP were followed, and cerebral perfusion pressure (CPP) calculated. The integrity of the autoregulation was estimated using a calculated Modified-Long Pressure Reactivity Index (mL-PRx). After stabilisation, hemodynamics and physiological parameters were similar and normal in both groups. At the end of the HS, ICP was 16 mmHg in NICPG vs. 32 in EICPG (p = 0.0010). CPP was 30 mmHg in NICPG vs. 6 mmHg in EICPG (p = 0.0254). After aorta occlusion CPP increased immediately in both groups reaching after 15min up to104 mmHg in NICPG vs. 126 mmHg in EICPG. Cerebrovascular reactivity seems to be altered during bleeding and occlusion phases in both groups with positive mL-PRx. The alteration was more pronounced in EICPG, but reversible in both groups. tREBOA is lifesaving by restoration the cerebral circulation defined as CPP in animals with HS with normal or elevated ICP. Despite the observation of short episodes of cerebral autoregulation impairment during the occlusion, mainly in EICPG, tREBOA seems to be an effective tool for improving cerebral perfusion in HS that extends the crucial early window sometimes known as the "golden hour" for resuscitation even after a traumatic brain injury.

Behavioral disorders in Parkinson disease: current view.

Patients with Parkinson disease (PD) frequently experience several behavioral symptoms, such as anxiety, apathy, irritability, agitation, impulsive control and obsessive-compulsive or REM sleep behavior disorders, which can cause severe psychosocial problems and impair quality of life. Occurring in 30-70% of PD patients, these symptoms can manifest at early stages of the disease, sometimes even before the appearance of classic motor symptoms, while others can develop later. Behavioral changes in PD show distinct patterns of brain atrophy, dopaminergic and serotonergic deterioration, altered neuronal connectivity in frontostriatal, corticolimbic, default mode and other networks due to a cascade linking molecular pathologies and deficits in multiple behavior domains. The changes suggest a multi-system neurodegenerative process in the context of a specific α-synucleinopathy inducing a variety of biochemical and functional changes, the neurobiological basis and clinical relevance of which await further elucidation. This paper is intended to review the recent literature with focus on the main behavioral disturbances in PD patients, their epidemiology, clinical features, risk factors, animal models, neuroimaging findings, pathophysiological backgrounds, and treatment options of these deleterious lesions.

Omics in IgG4-related disease.

Research on IgG4-related disease (IgG4-RD), an autoimmune condition recognized to be a unique disease entity only two decades ago, has processed from describing patients' symptoms and signs to summarizing its critical pathological features, and further to investigating key pathogenic mechanisms. Challenges in gaining a better understanding of the disease, however, stem from its relative rarity-potentially attributed to underrecognition - and the absence of ideal experimental animal models. Recently, with the development of various high-throughput techniques, "omics" studies at different levels (particularly the single-cell omics) have shown promise in providing detailed molecular features of IgG4-RD. While, the application of omics approaches in IgG4-RD is still at an early stage. In this paper, we review the current progress of omics research in IgG4-RD and discuss the value of machine learning methods in analyzing the data with high dimensionality.

Corrigendum: Stimulation of hair regrowth in an animal model of androgenic alopecia using 2-deoxy-D-ribose

Corrigendum: Stimulation of hair regrowth in an animal model of androgenic alopecia using 2-deoxy-D-ribose

A bibliometric and visualization analysis of global trends and frontiers on macrophages in abdominal aortic aneurysms research.

Macrophages are key regulators of the inflammatory and innate immune responses. Researchers have shown that aberrant expression of macrophages contributes to the development of abdominal aortic aneurysms (AAA). However, a comprehensive bibliometric analysis exploring the research status and knowledge mapping of this area is lacking. This study aimed to explore the research status, knowledge mapping and hotspots of macrophages in AAA research from a bibliometric perspective. In this study, we retrieved articles published between 2000 and 2022 on macrophages associated with AAA research from the Web of Science Core Collection (WoSCC) database. The retrieved literature data were further analyzed using Citespace and VOSviewer software. A total of 918 qualified publications related to AAA-associated macrophages were retrieved. The number of publications in this field has been increasing annually. China and the United States were the 2 main drivers in this field, contributing to more than 64% of the publications. In addition, the US had the most publications, top institutions, and expert researchers, dominating in research on macrophages in AAA. The Harvard University was the most productive institution, with 60 publications. The journal with the most publications was Arteriosclerosis, Thrombosis, and Vascular Biology (86). Daugherty Alan was the most prolific author (28 publications) and he was also the most cited co- author. Furthermore, the exploration of established animal models, macrophage-related inflammatory-microenvironment, macrophage-related immune mechanism, clinical translation and molecular imaging research remained future research directions in this field. Our findings offered new insights for scholars in this field. They will help researchers explore new directions for their work.

Intranasal administration of recombinant human BDNF as a potential therapy for some primary headaches

BackgroundIn addition to its critical role in neurogenesis, brain-derived neurotrophic factor (BDNF) modulates pain and depressive behaviors.MethodsIn a translational perspective, we tested the anti-migraine activity of highly purified and characterized recombinant human BDNF (rhBDNF) in an animal model of cephalic pain based on the chronic and intermittent NTG administration (five total injections over nine days), used to mimic recurrence of attacks over a given period. To achieve this, we assessed the effects of two doses of rhBDNF (40 and 80 µg/kg) administered intranasally to adult male Sprague–Dawley rats, on trigeminal hyperalgesia (by orofacial formalin test), gene expression (by rt-PCR) of neuropeptides and inflammatory cytokines in specific areas of the brain related to migraine pain. Serum levels of CGRP, PACAP, and VIP (by ELISA) were also evaluated. The effects of rhBDNF were compared with those of sumatriptan (5 mg/kg i.p), administered 1 h before the last NTG administration.ResultsBoth doses of rhBDNF significantly reduced NTG-induced nocifensive behavior in Phase II of the orofacial formalin test. The anti-hyperalgesic effect of intranasal high-dose rhBDNF administration in the NTG-treated animals was associated with a significant modulation of mRNA levels of neuropeptides (CGRP, PACAP, VIP) and cytokines (IL-1beta, IL-10) in the trigeminal ganglion, medulla-pons, and hypothalamic area. Of note, the effects of rhBNDF treatment were comparable to those induced by the administration of sumatriptan. rhBDNF administration at both doses significantly reduced serum levels of PACAP, while the higher dose also significantly reduced serum levels of VIP.ConclusionsThe findings suggest that intranasal rhBDNF has the potential to be a safe, non-invasive and effective therapeutic approach for the treatment of primary headache, particularly migraine.

Establishment of Animal Infection Model of Spirometra Mansoni and Identification of Spirometra Mansoni by Enzyme-Linked Immunosorbent Assay.

Objective: Spirometra mansoni is a crucial zoonotic parasite. Its larvae are more harmful than adult worms due to their ability to migrate through the host's tissues and organs. Therefore, it is necessary to establish an animal model of spargana for observing pathological changes and exploring diagnostic techniques. Methods: In this study, we infected Kunming mice and cats without any pathogens by feeding sparganum (with the scolex and neck) in order to understand the infection cycle of S. mansoni and explore the preservation host of sparganosis. The infection of S. mansoni was determined by fecal detection and enzyme-linked immunosorbent assay (ELISA). Results: In the model of cats, the eggs of S. mansoni were found in the feces ten days after the infection. The serum-specific IgG antibodies against S. mansoni were positive in experimental groups (mice and cats), and after sixty days, the S. mansoni worms isolated from experimental groups were collected. Conclusion: In conclusion, the experimental results show that mice and cats can be stably infected with S. mansoni through feeding sparganum (with the scolex and neck). The infection method of this study has the potential to establish a practical model for investigating the diagnostic process of S. mansoni, laying the groundwork for application and development. ELISA was used to diagnose mice and cats infected with sparganosis mansoni, providing a case for non-invasive identification of animal sparganosis.

Morphological characteristics of rabbits’ carpal tunnel: Evaluation of its potential for carpal tunnel research

This study aimed to define the main anatomic components and describe the normal size and form of the carpal tunnel in rabbits using imaging techniques. It was also aimed to evaluate the potential of rabbits as an animal model for carpal tunnel research. The forelimbs of eight adult New Zealand rabbits were investigated using histology, magnetic resonance, and computed tomography. Two cadavers were used for anatomical dissection to support the findings. The carpal tunnel was examined at proximal and distal levels, and it was found that the flexor retinaculum is two-layered in rabbits. Both vascular nerve bundles, including the median nerve and the ramus palmaris of the ulnar nerve, were within two layers of it and passed through the carpal tunnel. The deep digital flexor, superficial digital flexor, and radial carpal flexor tendons were observed within the carpal tunnel. The flexor pollicis longus tendon is not found in rabbits. The area of the carpal tunnel narrowed towards the distal end in three methods, and its depth decreased in CT and MRI. The length of the accessory carpal bone determined this depth. The study found that rabbits' carpal tunnels are morphologically similar to those of humans compared to dogs. This situation is thought to pave the way for more accurate approaches to carpal tunnel research in rabbits.

Xenon and Argon as Neuroprotective Treatments for Perinatal Hypoxic-Ischemic Brain Injury: A Preclinical Systematic Review and Meta-Analysis.

Xenon and argon are currently being evaluated as potential neuroprotective treatments for acquired brain injuries. Xenon has been evaluated clinically as a treatment for brain ischemia with equivocal results in small trials, but argon has not yet undergone clinical evaluation. Several preclinical studies have investigated xenon or argon as treatments in animal models of perinatal hypoxic-ischemic encephalopathy (HIE). A systematic review of MEDLINE and Embase databases was performed. After screening of titles, abstracts, and full text, data were extracted from included studies. A pairwise meta-analysis of neuroprotective efficacy was performed using a random effects model. Heterogeneity was investigated using subgroup analysis, funnel plot asymmetry, and Egger's regression. The protocol was prospectively registered on PROSPERO (CRD42022301986). A total of 21 studies met the inclusion criteria. The data extracted included measurements from 1591 animals, involving models of HIE in mice, rats, and pigs. The meta-analysis found that both xenon and argon had significant (P < .0001) neuroprotective efficacies. The summary estimate for xenon was 39.7% (95% confidence interval [CI], 28.3%-51.1%) and for argon it was 70.3% (95% CI, 59.0%-81.7%). The summary effect for argon was significantly (P < .001) greater than that of xenon. Our results provide evidence supporting further investigation of xenon and argon as neuroprotective treatments for HIE.

Chronic Hypoxia in an EXTrauterine Environment for Neonatal Development Impairs Lung Development.

Severe fetal hypoxia poses a significant risk to lung development resulting in severe postnatal complications. Existing chronic hypoxia animal models lack the ability to achieve pathologically reduced fetal oxygen without compromising animal development, placental blood flow, or maternal health. Using an established model of isolated chronic hypoxia involving the Extrauterine Environment for Neonatal Development (EXTEND), we are able to investigate the direct impact of fetal hypoxia on lung development. Oxygen delivery to preterm fetal lambs (105-110 days GA) delivered by cesarean section was reduced, and animals were supported on EXTEND through the canalicular or saccular stage of lung development. Fetal lambs in hypoxic conditions showed significant growth restriction compared to their normoxic counterparts. We also observed modest aberrant vascular remodeling in the saccular group after hypoxic conditions with decreased macrovessel numbers, microvascular endothelial cell numbers, and increased peripheral vessel muscularization. In addition, fetal hypoxia resulted in enlarged distal airspaces and decreased septal wall volume. Moreover, there was a reduction in mature SFTPB and processed SFTPC protein expression concomitant with a decrease in AT2 cell number. These findings demonstrate that maternally-independent fetal hypoxia predominantly impacts distal airway development, AT2 cell number, and surfactant production with mild effects on the vasculature.

Procurement and Preservation of Neonatal Porcine Cardiac Tissue.

The porcine and human heart are remarkably similar in cardiac physiology and biochemistry. Translational research involving the porcine biomedical model is becoming increasingly applicable for the study of human cardiac function in health and disease. Presently, few protocols exist for collecting experimentally viable cardiac tissue from large animal models, particularly during neonatal maturation. To address this deficiency, we have developed a technique to procure and preserve ventricular tissue from neonatal piglets at day 3 (n=4) and day 30 (n=6) post-partum. Piglets were subjected to a strict sedation, anesthesia, and analgesia regimen. During surgery, cardiopulmonary indices of electrocardiogram, heart rate, systolic and diastolic blood pressure, respiration rate, peripheral O2 saturation, and end-tidal CO2 were monitored continuously to ensure normal cardiac function. Prior to cardiectomy, each heart was perfused with an intravenous administration of heparin (10 ml/kg) and ice-cold Custodiol HTK cardioplegia solution (10 ml/kg). After cardiac explantation, myocardial samples (dimensions: 1 x 1 x 1 cm) were dissected from the left and right ventricles and snap-frozen in liquid nitrogen. Analysis via SDS-PAGE and densitometry demonstrated that myofibrillar proteins are stable and undegraded. Western Blots showed full expression of protein. These results suggest that the detailed cardiac tissue procurement technique preserves the experimental viability of the cardiac tissue and prevents the degradation of myofibrillar proteins.

Estimation of genetic parameters for reproductive traits in Korean dairy cattle.

In Korea, dairy cattle breeding programs have historically prioritized productive, conformation traits, leading to positive improvements, yet reproductive traits have lagged in development. This study was conducted to develop the breeding program of key reproductive traits in the Korean dairy cattle population. Utilizing data from 7,596 farms and over seven million observations, we conducted quality control to rectify manual entry errors and selected traits in line with international genetic evaluation standards. Traits analyzed included heifer conception rate (HCR), interval from calving to first insemination (CF), cow conception rate (CCR), interval from first to last insemination (FL), and days open (DO). Genetic parameters were estimated using a single trait animal model for HCR and a multiple lactation animal model for CF, CCR, FL, and DO, considering contemporary group of herd-insemination year, insemination month, and monthly age (MA) as fixed effects. Results showed low heritability estimates, ranging from 0.007 to 0.035 across different traits and lactations. Theoretical reliability appears to be low on average due to the influence of heritability, but it showed sufficiently high reliability in some sires (over 0.8). In terms of genetic and phenotypic trends, capacity for reproductive traits declined for a long time until around 2014. In recent individuals, improved trend can be found. This study addressed the critical need for enhancing reproductive efficiency to complement the existing breeding goals, thereby supporting sustained economic viability in the dairy industry. The results underscore the need for improved data quality and methodological adjustments for reproduction records to enhance the genetic evaluation of dairy cattle in Korea.

Primary Bone Tumors and Breast Cancer-Induced Bone Metastases: In Vivo Animal Models and New Alternative Approaches

Bone is the preferential site of metastasis for the most common tumors, including breast cancer. On the other hand, osteosarcoma is the primary bone cancer that most commonly occurs and causes bone cancer-related deaths in children. Several treatment strategies have been developed so far, with little or no efficacy for patient survival and with the development of side effects. Therefore, there is an urgent need to develop more effective therapies for bone primary tumors and bone metastatic disease. This almost necessarily requires the use of in vivo animal models that better mimic human pathology and at the same time follow the ethical principles for the humane use of animal testing. In this review we aim to illustrate the main and more suitable in vivo strategies employed to model bone metastases and osteosarcoma. We will also take a look at the recent technologies implemented for a partial replacement of animal testing.