Monomeric C-reactive protein (mCRP), once thought to be a figment of the imagination and whose biological activity was ascribed to its sodium azide preservative, has now pronounced itself as a critical molecule playing a direct role in mediating many of the acute and chronic aberrant pathological responses to inflammation. In this focused mini review, we describe the currently attributed pathobiological interactions of mCRP in disease, where its tissue and cellular distribution and deposition have recently been clearly characterized and linked to inflammation and other pathway-associated progression of neurological and cardiovascular complications and deleterious outcomes. and focus upon current opinions as to the diagnostic and prognostic potential of mCRP-plasma circulating protein and define the possible future therapeutics including ongoing research attempting to block CRP dissociation with small molecule inhibitors or prevention of cell surface binding directly using antibodies or modified orphan drug targeting directed towards CRP, inhibiting its cellular interactions and signaling activation. There is no doubt that understanding the full influence of the biological power of mCRP in disease development and outcome will be considered a critical parameter in future stratified treatment. The physiological functions of C-reactive protein (CRP) and the precise mechanisms through which it activates complement, modulates the innate immune system, and opsonizes invading pathogens are fully understood to date  and therThe blood insoluble mCRP, , is produced in inflammatory environments following lyso-phosphatidylcholine recognition and subsequent Fc-Gamma receptor/integrin binding with compatible ‘activated’ blood cells, platelets, circulating microvesicles and at sites of tissue damage . PotempaThis mCRP dissociated form is now understood to be capable of directly and actively aggravating perpetuating inflammation at least in part through activation of M1 macrophage phenotype and subsequent expression of cytokines including tumor necrosis factor-alpha (TNF-α) and IL-6, in addition to promoting platelet and platelet-macrophage adhesion and aggregation . Whilst cardiovascular disease. Development of atherosclerosis is a complex chronic condition, with many risk factors, occurring over several decades primarily during adult life  in atherosclerotic lesions of hypercholesterolemic rabbits as well as in unstable or ruptured human coronary artery plaques . Recentlet al.  where thRecently, Melnikov et al.  describeTaken together all these findings can be interpreted as providing evidence of a direct role of mCRP in promoting the chronic buildup of plaque in vessel walls, potential shifting the balance to an unstable/vulnerable and inflammatory phenotype, and provision of a systemic circulating micro-environment triggering thrombotic potential.neurological conditions mCRP also seems to make a significant and deleterious contribution to disease pathology. Pathological conditions involving neuroinflammation appear to encompass expression of or tissue accumulation of mCRP for example within the brain after stroke. More than a decade ago, Yu et al.  peptide {Aβ(42)}. When they examined the frontal cortex of individuals who died with Alzheimer’s disease (AD), they discovered the presence of mCRP in cortical Aβ plaques and suggested it may have a role in amplifying local neuro-inflammation and promoting tissue degeneration. Since then, further investigations have confirmed the ability of mCRP following hippocampal injection in mice, to induce behavioral and memory/cognitive deficits (which was protected against following co-injection of a specific antibody against mCRP) whilst co-localizing with both phosphorylated Tau (p-Tau) and Aβ(42); an in vitro capability of mCRP to induce Tau phosphorylation and stimulate production of other AD-precursors including presenilin enhancer protein-2 and phosphorylated amyloid precursor protein  block the binding and activity of mCRP on the cell surface or 3) ‘clear’ mCRP tissue deposits by solubilization, opsonization or phagocytosis; might have major clinical significance. So far, breakthroughs have been limited, however, Fujita et al. recently showed that mCRP-specific antibody clone 3C suppressed leukocyte infiltration and symptoms of rheumatoid arthritis in a proof-of-concept murine collagen antibody induced model as well as lupus nephritis symptoms in an MRL/MP-lpr/lpr lupus prone model  prevent the dissociation of nCRP to mCRP ne model . Similarne model , found tin vitro, and in vivo, went on to demonstrate a significant reduction in inflammation and tissue deposition of mCRP in a rat model of myocardial infarction in the presence of this small molecule. An X-ray crystal structure of the pCRP-bis(PC)-H drug complex showed that five palindromic drug molecules were required to bind to phosphocholine sites and this created a linkage between two pentamers through crosslinking and occlusion of the ligand-binding B face, preventing dissociation.An alternative approach is to identify or synthesize molecules with the ability to prevent nCRP dissociation mediated by phospholipase A2-dependent exposure to lysophosphatidylcholine at the surface of cell membranes. In this regard, Pepys et al. first demonstrated that 1, 6-bis(phosphocholine)-hexane was a small molecule capable of binding in a ration of 5:2 with individual sub-units of CRP; on binding, and applied to a rat model of myocardial infarction, both myocardial infarction volume and cardiac dysfunction were abrogated . Thiele in vitro evidence of prevention of mCRP-membrane binding with abrogation of inflammatory associated biology. Caution should be employed when considering therapeutic manipulation of a molecule such as CRP to ensure that the important normal physiological functions of the parent pentamer such as complement activation and opsonin protective characteristics remain. For this reason, the ideal therapeutic might be one that simply abrogates the dissociation thereby maintaining or in fact increasing the amount of circulating anti-inflammatory CRP whilst reducing the pro-inflammatory mCRP.This interaction abrogates binding of pCRP to known ligands such as LDL and blocks CRP-mediated complement C1q activation. Recently, Caprio et al.  and SlevCRP has now proven itself not only to be a marker of inflammation (in its native form) but more importantly, an important driver of the process and initiator of signal transduction cascades associated with pathologies linking to neurodegeneration, unstable atherosclerosis and autoimmune conditions amongst others. Modulation of mCRP might help protect from development of dementia, stroke and heart disease, mediated by chronic inflammatory-associated tissue damage. Over the coming years it would be anticipated that more pathologies harbor critical associations with mCRP deposition will be identified and characterized. Hence, strategies for therapeutic stabilization of native CRP thereby preventing its dissociation and/or CRP-binding proteins that block its cellular binding should be sought with urgency, whilst formulation of commercial technology enabling accurate measurement of this protein in the plasma should be prioritized.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: High-quality transitions of care are crucial for patient safety in hospitals, yet few undergraduate curricula include transition-of-care training. In 2012, the Wayne State University Office of Graduate Medical Education (WSUGME) required its residency programs to use the SAIF-IR mnemonic  to ensure accurate and uniform handoffs. Subsequent program evaluations indicated that resident awareness and adoption of the mnemonic at our primary clinical site, Ascension Providence Rochester Hospital (APRH), could be improved. According to our institution's 2016 Clinical Learning Environment Review (CLER), 88% of residents reported following a standardized transition of care handoff, and 53% reported that faculty rarely supervised their handoffs. A 2016 WSUGME internal survey also revealed low rates of awareness (7% to 10%) of the mandated mnemonic. WSUGME then created a direct observation tool, the Transitions of Care-Clinical Evaluation Exercise (TOC-CEX), for faculty to monitor resident skill in using the mnemonic and thus standardize transitions of care as a practice habit at APRH.Methods: Since 2014, WSUGME had relied on 2 methods for training residents in the required handoff mnemonic: (1) introduction to the SAIF-IR mnemonic during the WSUGME orientation for all interns and (2) simulations during an objective simulated handoff evaluation activity for all postgraduate year (PGY) 1s and PGY 2s. In 2017, WSUGME innovated a direct observation tool, the TOC-CEX, for adoption by faculty at APRH to assess resident knowledge of and monitor their skill in using the SAIF-IR mnemonic in 3 primary care programs. The total number of possible participants was 138, and the actual number of individuals in the sample was 95. A majority (86%) of the observations during the study period were of PGY 1 residents, and thus the analysis reflects the ratings of 99% of all interns but only 69% of all possible residents.Results: WSUGME found that faculty use of a direct observation instrument in the clinical learning environment during 2017-2019 increased awareness and adoption of the SAIF-IR mnemonic among residents. Using a z-test of equal proportions on resident responses on an internal WSUGME survey, we found a significant rise in the percentage reporting yes to the question “Does your program have a mechanism for monitoring handoffs?”  and in the percentage identifying SAIF-IR in response to the question “Does your program endorse a specific mnemonic for organizing the contents of a verbal handoff?” . The increase from 2016 to 2017 is the result of the implementation of the TOC-CEX in the interim .Conclusion: Our research found that use of the TOC-CEX to monitor resident handoffs resulted in improved awareness and adoption of the SAIF-IR mnemonic in the clinical learning environment. Program leadership reported that the practice was both feasible and well accepted by residents, faculty, and the APRH chief medical officer as the TOC-CEX became a customary component of APRH organizational culture and was perceived as central to quality patient care. That study, published in 2015, showed increases in residents’ awareness of the importance of accurate and thorough communication during handoffs but relied on self-reported data with no documentation of TOC use in the clinical setting.11In 2012, the Wayne State University Office of Graduate Medical Education (WSUGME) established a task force to explore TOC protocols and selected the SAIF-IR mnemonic  to ensure uniform and accurate handoffs.WSUGME's subsequent examination of data sources verifying the extent of residents’ adoption of the mnemonic was inconclusive, indicating that the desired goal of standardizing the use of the SAIF-IR mnemonic had not been fully achieved. According to our institution's May 2016 Clinical Learning Environment Review (CLER) site visit report, 88% of residents in group interviews reported following a standardized procedure for handling TOCs between shifts, and slightly more than half (53%) reported that faculty rarely supervised their shift-to-shift handoffs. During this visit, the CLER visitors reported that neither a standard mnemonic nor a written template was used during one observed handoff. In addition, resident responses to the annual WSUGME internal survey administered in fall 2016 revealed low rates of awareness (7% to 10%) of the specific TOC mnemonic required.13In this article, we detail our innovation for standardizing the use of the SAIF-IR mnemonic among primary care residents on inpatient services at our primary clinical site, Ascension Providence Rochester Hospital (APRH). Previous studies have confirmed the importance of providing residents with structured training about TOC mnemonic use via interventions such as bootcamps, didactic sessions, and/or simulated learning experiences, but little research is available on TOC postintervention reinforcement via direct observation of actual patient encounters.Our expectation was that use of a direct observation instrument, Transitions of Care-Clinical Evaluation Exercise (TOC-CEX), by faculty as both a monitoring and education tool would increase awareness and adoption of TOCs as a practice habit and contribute to culture change in the clinical learning environment.On April 3, 2020, the Wayne State University Institutional Review Board determined that this study (Protocol IRB-20-03-1951) was exempt from review.Located in southeast Michigan in the Metropolitan Detroit area, WSUGME provides administrative and curricular oversight of 7 residency programs with approximately 145 residents and 6 major hospital partners—the largest participating site being APRH, a 220-bed community hospital located in Rochester, Michigan, a suburb of Detroit. The study population included residents in 3 programs at APRH: internal medicine, family medicine, and transitional year. Using the TOC-CEX, faculty generated 223 observations of 95 residents, which took place at APRH between July 2017 and September 2019. During the 3 years studied, 138 unique residents were in the 3 programs, 96 of whom were interns and 42 of whom were postgraduate year (PGY) 2 and PGY 3 residents. A majority (86%) of the TOC-CEX observations were of interns, with the remainder (14%) being observations of PGY 2s and PGY 3s. Thus, the study sample reflects ratings of 99% of all interns during the study period but only 69% of possible residents.WSUGME used 3 data sources to gauge the percent change in resident awareness and adoption of the mnemonic: (1) CLER site visit reports submitted by an ACGME team of evaluators in 2016 and in 2018; (2) responses to 2 questions about handoff use on an annual graduate medical education survey distributed to residents each October across 4 years ; and (3) faculty observations of resident handoffs using the TOC-CEX from July 2017 through September 2019.14 Previous studies have found that simulation can be an effective method for teaching handoffs, particularly when accompanied by observer feedback and curricular requirements.15 Such deliberate practice allows residents to rehearse foundational skills across 2 yearly iterations in a low-stakes environment and also demonstrates the WSUGME commitment to policy monitoring while providing opportunities to refine handoff education.16 Beginning in summer 2017, WSUGME encouraged faculty adoption of the TOC-CEX as a direct observation tool  of each component of the mnemonic, and a numerical evaluation of the handoff as a whole (emerging competency/at expected competency/above expectations).The TOC-CEX was modeled after the Mini-Clinical Evaluation Exercise, part of a suite of clinical assessments recommended by the American Board of Internal Medicine.18WSUGME makes the TOC-CEX available to faculty as an electronic template via New Innovations, a widely used web-based residency management system. The WSUGME director of education downloads the completed instruments. The administration protocol followed by APRH faculty varies slightly according to specialty. In internal medicine, 4 teams that include a senior resident and 2 interns meet twice daily (morning and afternoon) to conduct handoffs; faculty monitor the handoffs and conduct a written direct observation of each resident 3 or 4 times per month. In family medicine and transitional year, the rounding faculty member that week observes a daily sign-out in the afternoon between senior residents and interns, selecting 1 resident per day, typically the resident who has seen the most patients, so that by the end of the week, all residents have been assessed using the TOC-CEX. As the family medicine associate program director noted, “We stick to a schedule and make the process very familiar to all residents from the start of their training, setting expectations” about the required mnemonic on which they will be assessed every week, an approach “which has decreased resistance.” The internal medicine program director commented, “Faculty oversight of the handoff process by direct observation using the SAIF-IR TOC-CEX ensures that residents are competent in communicating with team members in the handoff process. We [also] do multiple rehearsals” as a regular component of clinical training. As a result, “Most of our residents agree that medical errors could be reduced by ensuring that patient information on the handoff report is up to date and by conducting sign-outs in a more systematic manner.” These comments suggest how the TOC-CEX as both an instructional tool and monitored practice has become embedded in APRH organizational culture as a necessary and expected component of training and central to quality care, rather than just another administrative burden.19Analysis of the 3 data sources confirmed our expectation that faculty use of a direct observation instrument in the clinical learning environment during 2017-2019 would increase awareness and adoption of the SAIF-IR mnemonic among residents. By the time of the 2018 CLER site visit, 100% of APRH residents in group interviews reported using a standardized TOC, with 86% using a written template. CLER site visitors also observed appropriate SAIF-IR mnemonic use together with faculty supervision during 2 observed handoffs. Further, APRH percentages reporting faculty supervision are well above national norms. According to the 2018 CLER National Report, when asked how frequently attending physicians supervise their shift-to-shift handoff process, 23.3% of respondents nationwide said daily and approximately 15% said weekly, compared with 29% of APRH residents reporting daily and 57% weekly supervision.z-test of equal proportions showed a significant rise in the percentage reporting yes for question 1, “Does your program have a mechanism for monitoring handoffs?”  and in the percentage identifying SAIF-IR in response to question 2, “Does your program endorse a specific mnemonic for organizing the contents of a verbal handoff?” . The change is attributed to the increase from 2016, prior to TOC-CEX introduction, to 2017, after TOC-CEX implementation . No other year-to-year changes were significant.Most importantly, resident responses to questions about handoff use in the WSUGME internal survey in October of each academic year revealed significant increases in awareness of handoff monitoring across the study period . A z-tes, 181 of 219 (83%) as expected competency, and 31 of 219 (14%) as above expectations. Four TOC-CEXs were returned with missing data. WSUGME found that the overall quality ratings showed very high interrater reliability; the intraclass correlation coefficient (ICC) of mean ratings was ICC[2]=0.96. This result indicates strong interrater agreement about the quality of a TOC.With regard to adoption of the required mnemonic for handoffs, faculty reported that the SAIF-IR mnemonic was used in 218 of 223 observations (98%). With regard to the overall quality of the handoffs, faculty rated 7 of 219 (3%) as emerging competency18 Implementation of the TOC-CEX proved both feasible and well accepted by residents, faculty, and the APRH chief medical officer, as the new practice was incorporated with minimal or no disruption in patient care and reflected an ethic of collaboration and teamwork in service of patient safety while also reinforcing a commitment to policy adherence. Further, adoption of the TOC-CEX is part of WSUGME's continuous quality improvement approach mandated by the ACGME, a model that focuses on innovation and growth by promoting management tools such as performance dashboards and SMART-formatted  goals to transform the culture of GME. A core continuous quality improvement principle is that improvement requires change that must be monitored.20Adoption of the SAIF-IR mnemonic has been the core of TOC training in all our residency programs since 2012, but implementation of the TOC-CEX for faculty monitoring of resident use in the hospital site appears crucial. Via systematic participation in embedded clinical practices, residents observe faculty engagement in quality and safety, which is important because, as Myers and Nash argue, residents can become a “powerful force for changing hospital culture given their…natural ability to influence one another through social networks [and have] the potential to affect patient care outcomes through their knowledge, skills, and attitudes.”In terms of feasibility, WSUGME was able to address incoming interns’ lack of TOC preparedness without adding either a training burden  or substantial expenditure . With respect to training resources, WSUGME has access to a clinical skills center affiliated with our medical school that charges a fee for conducting the objective simulated handoff evaluations. However, any residency program could hold a similar TOC workshop at lower cost because no simulated patients are required. As explained earlier, in our training model, PGY 2s rate the skill of PGY 1s in using the SAIF-IR mnemonic during the objective simulated handoff evaluation.Limitations of this study include implementation in a single setting  and a small study population of internal medicine, family medicine, and transitional year residents across 3 years. In addition, adoption of a direct observation instrument such as the TOC-CEX by specialties like dermatology might be less suitable because physicians in these domains do not regularly engage in acute hospital-based patient care and focus principally on hospital consulting activity. At the same time, the instrument is sufficiently modifiable so that medical educators can adapt it for implementation in a variety of other settings.21 Such data will assist medical educators in determining the most effective handoff training approaches in the interest of optimizing patient safety.Future studies could address the piloting of a TOC-CEX instrument or innovation of a similar monitoring rubric for adoption in specialties other than primary care, perhaps in tandem with simulated exercises such as the objective simulated handoff evaluation, and compare outcomes across specialties. In addition, researchers might study TOC-CEX implementation by larger residency programs in other types of clinical learning environments, perhaps an urban hospital with a high patient volume and acuity. Although we describe one approach to reducing possible resident resistance to handoff monitoring, the exploration of other strategies  warrants investigation. Finally, WSUGME agrees with Rosenbluth that in subsequent TOC research, the “ideal data collection instrument would also include outcome measures…such as medical errors and adverse events, [which are] more difficult to document but also provide more valuable data about the impact of [TOC] curricula.”Faculty employment of the TOC-CEX in the clinical setting appears key to ensuring adoption by residents programwide. As a result, WSUGME has been able to standardize the SAIF-IR mnemonic as an integral component of the primary care programs at our main teaching hospital.


Periodic breathing is a respiratory phenomenon frequently observed in patients with heart failure and in normal subjects sleeping at high altitude. However, until recently, periodic breathing has not been studied in wakefulness and during exercise. This review relates the latest findings describing this ventilatory disorder when a healthy subject is submitted to simultaneous physiological (exercise) and environmental  or pharmacological (acetazolamide) stimuli. Preliminary studies have unveiled fundamental physiological mechanisms related to the genesis of periodic breathing characterized by a shorter period than those observed in patients (11~12 vs. 30~60 s). A mathematical model of the respiratory system functioning under the aforementioned stressors corroborated these data and pointed out other parameters, such as dead space, later confirmed in further research protocols. Finally, a cardiorespiratory interdependence between ventilatory oscillations and heart rate variability in the low frequency band may partly explain the origin of the augmented sympathetic activation at exercise in hypoxia. These nonlinear instabilities highlight the intrinsic “homeodynamic” system that allows any living organism to adapt, to a certain extent, to permanent environmental and internal perturbations. Cardiovascular and respiratory systems are two distinct systems fulfilling different and complementary missions. Hence, to meet the metabolic demands of the body, they have to function together, as a whole, through multiple and complex cardiorespiratory coupling. Moreover, they must adapt their own functioning and their relationship when the organism is confronted by environmental and/or physiological stressors. These adaptations may lead to transient or chronic oscillatory outputs in both systems, observed in normal subjects or in patients. Recent technological and mathematical advances allow a better characterization and a fine-tuned understanding of these oscillatory physiological behaviors. The present work, ensuing successive protocols carried out by our research group, aims to review these specific mechanisms occurring in healthy humans exercising in hypoxia and other various stressors, such as hypercapnia and hyperoxia.2). Additional parameters may also be added, such as the frequency of the peak in each HRV band.Heart rate variability (HRV) is now a widely used tool to provide valuable information about cardiorespiratory status and health in a large range of subjects, from elite athletes  to heartPhysiological significations of HRV bands are yet to be totally comprehended. For example, ULF band is not fully understood but might be driven by complex interactions between circadian rhythm, core body temperature, metabolism, and the renin-angiotensin system , and is As HRV represents a key tool in assessing numerous parameters in healthy and pathological conditions, there is growing evidence about the relevance of analyzing its ventilatory counterpart. For example, periodic breathing and/or ventilatory disorders are frequent in chronic heart failure (CHF) patients , and perllations .These observations point out the key role of peripheral chemoreceptors in the genesis of breathing instability, triggered by simultaneous environmental (hypoxia) and physiological (exercise) stressors. It is also interesting to note that an increase in cardiac output (during exercise) exacerbates the oscillations. In contrast, in CHF patients, the predominant hypothesis to explain the Cheyne-Stokes respiration (CSR) pattern is an increase in circulation time delay due to low cardiac output .If a hypoxic environment can destabilize the breathing control system during mild exercise, what are the effect(s) of other environmental and pharmacological stimuli?2 (HCVR)?Chemosensitivity to hypoxia underlines the role of peripheral chemoreceptors (mainly carotid bodies) in ventilatory oscillations, but what about the contribution of central chemoreceptors, illustrated by the ventilatory response to CO2 inhalation during sleep and exercise considerably diminishes apnea occurrences in CSR by keeping CO2 arterial pressure above the apneic threshold . Th. Th2 or 2 (PaCO2) combined with a higher 2 from falling below the apnea threshold and the subsequent genesis of a central apnea cycle [2 inhalation. Whereas hypercapnia and added dead space reduce 2 above apnea threshold, the subsequent augmented Except environmental or pharmacological interventions, an artificial modification in upper airways might impact the breathing control system, for example, by adding an external dead space. This method has proven to be very efficient in drastically reducing periodic breathing in patients ,43,44 anea cycle . In healea cycle . The invThe mechanisms involved in ventilatory oscillations in healthy subjects at exercise in various environmental and/or pharmacological conditions are illustrated in eceptors , and, toeceptors . The per2 and CO2 [2 or CO2 inhalation [The successive attempts of modeling the ventilatory control system were based on the mass balance equation for O and CO2 . Whereas and CO2 ,53. Mode and CO2 . These shalation .2 and CO2 sensors , O2 and CO2 chemoreceptor gains, blood convection delay from lungs to chemoreceptors  and in subjects exposed to altitude, during sleep. The recent advances in fundamental knowledge helped to partially explain the complex interactions between respiratory and cardiovascular systems in normal subjects submitted to several stimuli (hypoxia and exercise). The observed oscillations in both signals may further illustrate the necessary adaptations to a new context, at a cellular and organism level, not too distant from the basal environment (as in CHF-related sleep apnea). A deeper reflection would even lead us to consider that variability/instability accommodation represents a crucial ability to maintain homeostasis in any new circumstances . As an e


Imbalance of Aβ and tau protein production and clearance are the key factors among many causes of Alzheimer's disease that leading to neurons degeneration and cognitive disorders. As a novel approach, glymphatic system quickly clear metabolic waste  from cerebral environment, and dysfunction of glymphatic system may relate to occurrence of Alzheimer's disease. Microinfarct is a common histopathologic situation occurring in aging brain and leads to dramatic increase the generation of metabolic by-product after neuronal injury, hindering the operation of glymphatic system and suppress cerebral spinal fluid (CSF) and cerebral interstitial fluid  exchange. Microinfarcts destruct the integrity of microvascular and microstructural tissue, result in Aβ deposition and tau phosphorylation that form neurofibrillary tangles and associated with the cause of Alzheimer's disease. Currently, it has been found that glymphatic system is involved in the pathological process of Alzheimer's disease. Improving the function of glymphatic system after cerebral microinfarcts could be developed as a new approach for Alzheimer's disease prevention and treatment. In this review, we will provide in-depth discussion on functional changes of glymphatic system after cerebral microinfarcts, further reveal pathogenesis of Alzheimer's disease and provide a potentially more effective method for treatment of Alzheimer's disease. Cerebral microinfarct is a common pathological event in patients with dementia and other cerebrovascular diseases Glymphatic perfusion of the central nervous system (CNS) serves multiple purposes in neurophysiology. It is important for delivery of nutrients, specifically glucose Aqp4 knockout mice compared with wild-type mice Alzheimer's disease (AD) is associated with the accumulation of toxic aggregates of Aβ and hyperphosphorylated tau in the brain. The concentration-dependent aggregation of Aβ peptides is determined by the balance between production and clearance mechanisms. Aβ peptides are generated from the amyloid precursor protein. Aβ can be cleared from the brain by several mechanisms, including intracellular and extracellular degradation, accumulation in plaques, transcytosis across the blood-brain barrier, and bulk clearance into the CSF. Unlike Aβ, tau is an intracellular protein, hyperphosphorylated tau is thought to be generated in the late stages of neurodegeneration in AD. The spread of tau follows the pattern of neuronal connectivity; therefore, clearance of tau from the brain is much slower than that of Aβ. AQP4-dependent glymphatic pathway for driving the removal of soluble Aβ from the interstitium is an important clearance system. In mice, Aβ is cleared along perivascular, and Aβ clearance was reduced by 55-65% in As a key component of the central lymphatic system, glymphatic system plays an important role in Alzheimer's disease (AD), brain injury (TBI), microinfarcts and other cognitive disorders. Here, we will discuss whether the pathogenesis between neurodegenerative diseases (such as Alzheimer's disease) and cerebral microinfarcts are caused by changes in glymphatic function after microinfarcts. This complex mechanistically glymphatic system could provide new therapeutic targets for neurological disorders associated with immune malfunction and protein aggregation, leading to new therapies.Microinfarcts are a common feature existing among elderly patients, particularly in those suffering mild cognitive dysfunction, vascular or Alzheimer's dementia and are thought to contribute to cognitive decline in elderly patients In addition to causing local tissue damage, microinfarcts can also lead to neurodegenerative changes in distal brain regions Data compiled from studies suggest that microinfarcts occur throughout brain, including cortex and subcortical gray and white matter. All cortical and subcortical areas are found to have microinfarcts by comparing incidence of lesions in cortical and subcortical areas. The microinfarcts are then assessed and provide a more complete description of microinfarcts in gray and white matter. The result of two studies specified that no difference was found in appearance of microinfarcts between cortical and subcortical regions, while another study showed that microinfarcts were more common in subcortical regions. However, whether microinfarcts occur preferentially in cortex or subcortical areas was not discussed in most of the studies. Regarding to specific brain areas, cerebral microinfarcts are most common in parietal and occipital areas. Others are only in cortical gray matter. White et al. Thus, these results suggest that microinfarcts can occur in all brain regions. However, the occurrence of lesions in different brain regions was not compared specifically, but some studies found microinfarcts were more abundant in cerebral cortex.Various underlying causes led to microinfarcts and were able to co-exist in a patient. Three of the main causes are cerebrovascular disease , microemboli and hypoperfusion. Researches on neuropathology discovered that cerebral microinfarcts are often accompanied by occurrence of severe cerebral amyloid vascular disease Neuropathologic studies further explored the relationship between cerebral microinfarcts and cerebral amyloid angiopathy. A study with brain examination of about 1000 community residents, it was found that cortical microinfarcts were accompanied by cerebral amyloid angiopathy Besides cerebral amyloidosis, cerebral microinfarcts have been found accompany with other small vascular diseases by MRI. Cerebral microinfarcts co-occur simultaneously with lacunar infarcts and were tied in with higher white matter volume and higher cerebral atrophy A 3T MRI study indicated that cerebral microinfarcts were not associated with diabetes or hypertension, but were significantly associated with hyperlipidaemia Neuropathologic studies have verified the association between cerebral microinfarcts and dementia and cognitive impairment, but the relationship between cerebral microinfarcts and other concurrent pathologies are complex and might require further investigation Altogether, these in-vivo MRI studies support neuropathological findings that cerebral microinfarcts are related to poor cognition and are not affected by other age-associated pathologies. However, it is unclear whether these associations are causal or not. Because cerebral microinfarcts are widely distributed throughout the brain, these lesions might cause enough disruption leading to functional impairment, which has been supported by animal studies Cerebral arterial circulation consists of anterior cerebral circulation supplied by internal carotid artery, vertebral artery and posterior cerebral circulation. The anterior circulation consists of the middle cerebral artery and the anterior artery, which are connected to posterior circulation through basilar artery, and the posterior cerebral artery by anterior and posterior communication arteries of the Circle of Willis Blood enters post-capillary venules from the cerebral capillaries, where the basement membranes of endothelial cell and astrocytes expands and provide a space around vessels for CSF drainage. Generally, blood from interior of the brain flows into larger central/deep veins and out of the cerebral cortex and subcortical white matter through cortical veins that extend to brain surface Recent studies have shown continuous exchange of cerebrospinal fluid and interstitial fluid (ISF). CSF enters the Virchow-Robin spaces from subarachnoid space through a combination of arterial pulse respiration and CSF pressure gradient. The subsequent transport of CSF into dense and complex brain parenchyma is driven by AQP4 water channels. The AQP4 channel is expressed in a highly polarized manner in astrocytic end-feet, which opens up brain vasculature Two-photon microscopy was applied to characterize the dynamics of the glymphatic system in mice by injecting fluorescent tracer labeled CSF into a large cisterna Therefore, we postulate that central nervous system (CNS) is the only organ system that lacks lymphatic vessels to assist in the removal of interstitial waste. Recent studies have identified the glymphatic system, a glia-dependent perivascular network that assists lymphatic function in the brain In an experiment with Oa-45 tracer injected into the lateral ventricle observed tracer flow from ISF to CSF through glymphatic system, and further transferred to neck lymph nodes and into blood, indicating that extracellular solute of brain cells can be cleared out of the brain through glymphatic system. Amyloid β (Aβ) injected into the brain of mice can be rapidly cleared by glymphatic system Entry of CSF through the perivascular space is essential for glymphatic ISF-CSF exchange and clearance function. Glymphatic transportation of CSF along the periarterial space, then the convective flow along the brain parenchyma, and outlet of interstitial fluid (ISF) along the perivenous space to neck lymphatic system is an energy-demanding process driven by a variety of mechanisms. The choroid plexus constantly produces CSF, creating a pressure that indicates the direction of fluid flow through ventricular system to subarachnoid space. What drives CSF into the pial along the perivascular space? Particular to arteries, pulsation produced by the smooth muscle cells generate impulses along the entire length of the pial meninges arteries and penetrate from the cortical surface into the perforating arteries of brain. Dobutamine, an adrenergic agonist, significantly increased pulsatile effect of mice and caused a flood of cerebrospinal fluid into parenchyma. Internal carotid artery ligation inhibited the pulsation in the opposite effect. In addition, reduction of pulse waves reduced the exchange of CSF-ISF AQP4, which is highly polarized and expressed on the end-feet of astrocytes, acts as a barrier between glymphatic system and brain parenchyma and plays a crucial role in regulating fluid flow between astrocytes, CSF and blood vessels In glymphatic system, ISF and CSF converge in brain parenchyma. During sleep, the intercellular space of brain cells increased by more than 60%, and the convection exchange between ISF and CSF also increased significantly, which accelerated the clearance of Aβ in CSF and made the concentration of Aβ in CSF during sleep lower than that during awake Body position can affect exchange efficiency of CSF and ISF through the change of hemodynamics, thus affecting glymphatic system. It was found that the material exchange efficiency of CSF and ISF was the highest in the right lying position and the lowest in the prone position. The reason may be that the heart position is higher in right lying position, which can promote blood suction and venous return, increase cardiac output, improve cerebral artery pulsation and thus improve the material exchange efficiency of CSF and ISF; while in prone position, the head is relatively higher, CSF outflow along neck vessels increases, which can reduce CSF in the brain, resulting in decreasing of the material exchange efficiency of CSF and ISF In the glymphatic system, PVS surrounds cerebral artery, CSF or CSF-ISF mixture flows in PVS, and cerebral artery pulsation can promote rapid movement of solute in PVS in the form of mixing and diffusion, thus completing the material exchange between CSF and ISF Microinfarcts destroy the integrity of microvessels or microstructures, reduce exchange of substances between CSF and ISF, promote Aβ and high phosphorylation of tau proteins deposits to form neurofibrillary tangles, and increase the risk of dementia. Dysfunction of glymphatic system leads to inflammatory responses, neurodegeneration, and cognitive impairment The core of a cerebral microinfarct is filled with microglia and surrounded by reactive astrocytes. AQP4 highly polarized and expressed on astrocytes terminal feet, is the barrier between glymphatic system and brain parenchyma and plays an important role in regulating astrocytes and the flow of fluid between CSF and blood vessels It has been reported that deletion of AQP4 gene leads to damaged posttraumatic glial scar formation and affects migration of astrocytes in primary cell culture Microinfarcts are common features in elderly patients, particularly those suffering mild cognitive decline or Alzheimer's disease, and some are thought to play a crucial role in age-related cognitive decline. However, the time course of individual development remains obscure because the microscopic infarcts are typically first identified only upon post-mortem examination. Microinfarcts described in the human clinical are incomplete and diffuse, lesion cores were associated with CD68-positive microglia and GFAP+ astrocyte processes, were typically microscopic (<1mm). GFAP-immunoreactivity and AQP4 polarization within perivascular end-feet, suggesting that water homeostasis and edema clearance may be compromised for a long period of time within a wide region surrounding these diffuse ischemic lesions. AQP4, which is localized to astrocytic end-feet, plays a role in CSF-ISF exchange and interstitial solute clearance through perivascular drainage pathways. The vascular polarization of astrocytic AQP4 is partly lost in reactive astrocytes in diffuse microinfarction brains. The finding that β-amyloid accumulation after microinfarcts was associated with loss of perivascular AQP4 polarization, and that presence of cortical parenchymal AQP4 is associated with CSF-ISF exchange, the microinfarcts related decline in glymphatic function might be in part attributable to dysregulation of astroglial water transport.There is increasing evidence that reduction of microinfarcts in glymphatic clearance contribute substantially to the accumulation of protein aggregates. Glymphatic system, as a metabolic waste discharge pathway and material delivery system of brain, plays an important role in CSF circulation by promoting the production of CSF and keeping the perivascular space unobstructed which leads to brain homeostasis. Polarizing distribution of AQP4 increase cell gap of brain, improve function of glymphatic system, which is expected to be the target of promoting the clearance of Aβ and tau protein. After microinfarcts, astrocyte activation, AQP4 polarity distribution disappeared, impaired glymphatic pathway function causes a wide range of neural and network damage, the disruption of solute transport surrounding these lesions may similarly drive outsized changes in neuronal network function because of the close association of deep lesions with the underlying white matter. The failure of glymphatic system clearance in microinfarcts might lead to accumulation of β-amyloid and hyperphosphorylated tau proteins and render the brain more vulnerable to developing neurodegenerative pathology or escalate the progression of cognitive dysfunction.More recent studies have revealed that β-amyloid and fibrillary tangles of tau in Alzheimer's disease are present in the interstitial fluid and CSF. The production and turnover of β-amyloid is strikingly rapid in humans. β-amyloid is not only produced by neurons. in fact, all cells produce β-amyloid. Bulk clearance by the glymphatic system might provide necessary and sufficient removal of extracellular β-amyloid until the end-of-life which time failure in adequate CSF bulk flow leads to accumulation of β-amyloid. This suggests that low activity of the glymphatic system could be a major risk factor for development of neurodegenerative diseases. Cerebral microinfarcts activated astrocytes resulting in loss of AQP4 which might reduce glymphatic CSF influx and the stagnation of CSF influx might accelerate β-amyloid accumulation. In turn, a spiral of protein accumulation, deformation of glymphatic routes and further reduction in protein clearance and pathology.Glymphatic dysfunction characterized by a failure of interstitial solute clearance is a central feature of aging brain, as well as CNS diseases including Alzheimer's disease, TBI and microinfarcts. While much is known about the regulation of glymphatic pathway, including the roles of cerebral arterial pulsatility, state of consciousness, and head position. There are no glymphatic therapies to intervene in these disease processes at present. The primary assignment of future studies will be identification of a target for regulation CSF-ISF exchange within the glymphatic pathway, to promote improved solute clearance in these diseases.


The blood–brain barrier (BBB) is a highly selective and restrictive semipermeable network of cells and blood vessel constituents. All components of the neurovascular unit give to the BBB its crucial and protective function, i.e., to regulate homeostasis in the central nervous system (CNS) by removing substances from the endothelial compartment and supplying the brain with nutrients and other endogenous compounds. Many transporters have been identified that play a role in maintaining BBB integrity and homeostasis. As such, the restrictive nature of the BBB provides an obstacle for drug delivery to the CNS. Nevertheless, according to their physicochemical or pharmacological properties, drugs may reach the CNS by passive diffusion or be subjected to putative influx and/or efflux through BBB membrane transporters, allowing or limiting their distribution to the CNS. Drug transporters functionally expressed on various compartments of the BBB involve numerous proteins from either the ATP-binding cassette (ABC) or the solute carrier (SLC) superfamilies. Pathophysiological stressors, age, and age-associated disorders may alter the expression level and functionality of transporter protein elements that modulate drug distribution and accumulation into the brain, namely, drug efficacy and toxicity. This review focuses and sheds light on the influence of inflammatory conditions and diseases such as Alzheimer’s disease, epilepsy, and stroke on the expression and functionality of the BBB drug transporters, the consequential modulation of drug distribution to the brain, and their impact on drug efficacy and toxicity. The blood–brain barrier (BBB) is a complex cellular barrier composed of a tightly sealed monolayer of specialized brain capillary endothelial cells lined on a basement membrane and surrounded by adjacent perivascular astrocytes, pericytes, and microglia. The major function of the BBB is to separate the circulating blood from the brain extracellular fluid and maintain homeostasis in the central nervous system (CNS) .2, a total length of capillaries of ~400 miles, microcapillaries with a diameter of 7–10 µm, and an average inter-capillary distance of ~40 µm ..13].Passive diffusion is the movement across a concentration gradient from high to low concentrations, without expenditure of biological energy or involvement of a carrier protein . PassiveThe second mechanism is endocytosis, which is defined as the vesicular transport across the BBB through receptor-mediated, adsorptive, or bulk-phase endocytosis ,18. ReceThe third mechanism is carrier-mediated transport, through which the solute molecules move from high to low concentrations across the BBB. This membrane-bound protein-mediated transport system is an energy-independent system that could be uniporter , symporter , or antiporter . This type of transporter is of utmost importance for CNS access to essential nutrients such as glucose through the glucose transporter 1, GLUT1 (SLC2A1) . CentralThe fourth mechanism is active transport across the BBB through a carrier protein. This process is energy-dependent and often coupled with ATP hydrolysis; it enables the movement of substances against their concentration gradient . There aWhile the four mechanisms are essential for maintaining brain homeostasis, the transport proteins expressed on the luminal (blood-facing) and abluminal surface of the BBB magnify the protection provided and strictly regulates the transcellular transport. Unfortunately, this process is affected by aging and age-associated disorders . Both thThe gatekeepers of the BBB are the endothelial cells lining its vasculature that express a variety of transporters that strictly regulate the influx and efflux of endogenous nutrients and exogenous toxins including xenobiotics and drugs . Below, The ATP-binding cassette (ABC) superfamily of transporters is perhaps the most studied family of active transporters that exists in nearly all living organisms ,25. The MDR1), ABCG2 , and ABCCs ; they are all efflux transporters, limiting therapeutic drug entry into the brain and preventing successful response to several agents intended to treat neurological disorders. Moreover, decreased expression or function of these transporters has been described in several CNS disorders .,41.40,41ABCG2 is expressed on the luminal side of BBB capillary endothelial cells and acts as an ATP hydrolysis-dependent efflux transporter of a wide range of structurally and chemically distinct compounds, including physiological substrates such as sulfate and glucuronide conjugates of estrone and dehydroepiandrosterone . In addiThe ABCCs are ~190 kDa proteins whose physiological functions include organic anions and nucleotide-based analogs transport, signal transduction, and toxin secretion . Althougβ-glucuronide), and glutathione disulfide (GSSG) -verapamil have initially assessed the function of Abcb1 activity at the BBB [11C]-verapamil concentration and binding to various sections of the brain is associated with decreased expression or function of the BBB. A recent clinical PET study compared the activity of ABCB1 at the BBB in AD patients to healthy subjects. The results of this study revealed a compromise transport activity of ABCB1 and suggested that ABCB1 downregulation might contribute to the progression of Aβ deposition in the brain; they observed a 23% increase in binding potential of [11C]-verapamil in the cortical region and higher binding potentials in other smaller brain regions of AD patients -verapamil efflux and ABCB1 activity was significantly reduced in AD patients Aβ into the brains of Abcb1a/b-/- double knockout (Abcb1-null) mice and wild-type controls -verapamil as a probe substrate for ABCB1 activity, Feldmann et al. studied the plasma-to-brain transport rate constant in a group of patients with drug-resistant mesial temporal lobe epilepsy and patients with adequate seizure control using medications [Epilepsy is a chronic neurological condition associated with excessive neuronal discharges resulting in spontaneous seizures observed clinically or recorded on electroencephalograms. Around 30% of patients with epilepsy experience resistance to available therapeutic options and continue presenting seizures . These oications . Compareications . A signiMounting evidence in epilepsy suggest that a high degree of mechanistic complexity and multiple pathways drives the pathological alterations observed. The impact of epilepsy on transporters other than the ABC family also showed an abnormal expression pattern. Using immunogold electron microscopy and analysis of epileptic patients undergoing cortical resection, Cornford et al. provided evidence that the glucose transporter SLC2A1 is downregulated in regions within and surrounding the seizure focus . EarlierAntiepileptic drugs such as carbamazepine are substrates of drug transporters such as ABCB1 and ABCG2. Although upregulation of these transporters has been demonstrated in clinical studies before, it is not known whether this leads to reduced anti-seizure drug concentrations in interstitial fluid. Rambeck et al. attempted to determine concentrations of antiepileptic drugs in serum, subarachnoid cerebrospinal fluid (CSF), and extracellular space (ECS) of epileptic tissues . SpecifiFollowing the notion that increased expression of transporters may result in decreased distribution of anti-seizure medications to the brain, it is logical to propose that inhibiting drug transporters can help increase brain accumulation of anti-epileptic drugs. Accordingly, Dickens et al. used the ABCB1 inhibitor tariquidar to determine whether it could modulate the transport of phenytoin, levetiracetam, and/or carbamazepine in seven different cell systems. Results obtained in all seven cell lines demonstrated that neither lamotrigine nor carbamazepine are substrates of ABCB1. Phenytoin showed characteristics of a weak substrate in polarized kidney cells (MDCK and LLC-PK1); however, it was not a substrate in any other cell models . SubsequCC genotype at ABCB1 3435, (impaired function) may be associated with drug resistant epilepsy [ABCB1 3435 position with anti-seizure drug response. These authors did not find a significant relation between the CC genotype and drug non-responders (drug resistant epilepsy) [Although these preclinical data were encouraging, similar results have not been reported in patients. An attempt to relate ABCB1 activity to antiepileptic drugs’ efficacy has been made using pharmacogenomic studies. It was initially reported that the epilepsy . Sills epilepsy) . Thus, tpilepsy) ,149.The impact of drug transporter-induced pharmacoresistance in epileptic patients could not be only limited to antiepileptic drugs. For instance, in patients with brain tumors , the presence of epilepsy is considered the most significant risk factor for chronic disability ,151. TheGlobally, stroke is the second leading cause of death and a major cause of serious long-term disability ,155. AboABCB1 (MDR1) gene expression and increased ABCB1 protein expression through activation of hypoxia-inducible factor alpha (HIF-1α) and nuclear factor κB [Further studies described below have tried to correlate upregulation of transporters to drug delivery into the ischemic brain and suggested a potential signaling pathway that induced the upregulation ,163. Foractor κB ,163. Spuactor κB . In theiactor κB . They foactor κB .Another family of transporters that may have utility in delivering neuroprotective drugs across the BBB is the SLCO family. In rodent brain, Slco1a4 is the primary drug transporting Slco isoform expressed at the BBB . In rat Amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, is a progressive neurodegenerative paralytic disease characterized by degradation and gliosis of the upper and lower motor neurons in the brain and the spinal cord, respectively . The etiIn the mid-1980s, clinical studies reported that ALS patients had an altered BBB integrity and permeability ,181,182.Riluzole (RLZ), one of the two FDA-approved drugs for ALS management, is a substrate of ABCB1 and ABCG2 ,193. A sMultiple sclerosis (MS) is a chronic inflammatory autoimmune CNS disease that cause demyelination, axonal loss, and neurodegeneration . MS is eSimilar to chronic neuroinflammation, MS compromises the integrity and function of the BBB. Evidence suggests that dysfunctional transporters at the BBB have been implicated in MS; however, the exact mechanism is uncertain. Several studies identified an important role for ABCB1, ABCG2, ABCC1, and ABCC2 in neuroinflammatory processes underlying MS pathology ,201,202.As the body’s central processing unit, the brain is equipped with a very efficient and complex firewall system named the BBB. In this review, we discussed the critical role of the BBB with a major focus on transporter proteins and their central physiological function in preserving cerebral homeostasis. The interplay between physiology and pathophysiology of transporters in the human brain has gained much interest in recent years. Although the role of the BBB drug transporters in regulating distribution of therapeutics in the CNS has been known for several decades, it is becoming increasing clear that BBB transporters are involved in the onset and progression of several neurological diseases (major findings are summarized in Recent research and neuroimaging studies greatly advance our understanding of BBB drug transporters, making them potential targets for several neurological diseases. Our awareness has also heightened about the impact of BBB dysfunction in the pathology of a wide range of CNS diseases; this provides invaluable insights into targeting BBB transporters as a way to prevent or slow the progression of certain neurological diseases. At the same time, it remains challenging to understand the complex physiological functions of these transporters. From many examples discussed in this review, various neurological diseases can have two completely different effects on the activity and expression of BBB transporters. Therefore, a better understanding of molecular and cellular mechanisms underlying function and expression and of these transporters will significantly improve the way we think about brain diseases and could lead to the discovery of novel BBB targets and approaches for therapeutic solutions.


GABBR1, which codes for the gamma aminobutyric acid receptor (GABAB1), and KCNN2, which codes for calcium-activated, potassium ion transporting SK2 channels responsible for membrane repolarization after cholinergic binding/signal transmission events. Cholinergic signal transmission pathways related to these proteins were examined in the Pathway Studio environment. The ion transport ontological associations indicated feasibility of a dietary choline support as a low-risk therapeutic intervention capable of modulating cholinergic sensory signaling in autism. Further research at the intersection of dietary status and sensory function in autism is warranted.Food is the primary human source of choline, an essential precursor to the neurotransmitter acetylcholine, which has a central role in signaling pathways that govern sensorimotor functions. Most Americans do not consume their recommended amount of dietary choline, and populations with neurodevelopmental conditions like autism spectrum disorder (ASD) may be particularly vulnerable to consequences of choline deficiency. This study aimed to identify a relationship between ASD and cholinergic signaling through gene set enrichment analysis and interrogation of existing database evidence to produce a systems biology model. In gene set enrichment analysis, two gene ontologies were identified as overlapping for autism-related and for cholinergic pathways-related functions, both involving ion transport regulation. Subsequent modeling of ion transport intensive cholinergic signaling pathways highlighted the importance of two genes with autism-associated variants:  Sensory processing dysfunction, which is commonly experienced by persons diagnosed with autism spectrum disorder, extends across auditory, visual, pressure, temperature, pain, vestibulary, and interoceptive processing domains . Some feWhile humans can make small amounts of choline endogenously, the replenishment of the pool of acetylcholine available in the human body depends heavily upon dietary choline intake . In the To explore the potential relationship between autism spectrum disorders (ASDs) and choline intake, we conducted an enrichment analysis of gene sets associated with ASD and cholinergic pathways, and constructed the model reflecting this relationship. Results of our study highlight the potential impact of dietary choline deficiency upon cholinergic signaling within the genetic context of autism.1 between December 2019 and March 2020. The Pathway Studio database contains functional relationships and pathways of mammalian proteins, including human, mouse, and rat genes. It contains over 1.4 million entities of 14 well-defined categories, including cells, proteins, disease, and small molecules, and more than 13.4 million relationships among these entities. The database includes over 24 million PubMed abstracts and 3.5 million Elsevier and third party full-text papers. Using the natural language processing (NLP) functionality of Pathway Studio, intersections of these gene sets’ pathways were rendered graphically, with connections indicating directional relationships being supported by the current scientific literature. This exploratory analysis and figure development which employed in part some of Pathway Studio’s graphical capabilities took place between March 2020 and November 2020. Since many of the autism-associated genes in the GWAS study have been recently identified as autism-associated only within the past 2 years, this gene/pathway modeling effort was supported by secondary analyses, including use of predictive tools in splice site identification.This study has focused on gene ontologies cataloged in the Gene Ontology (GO) database for enrichment analysis of an overlap between two gene sets, one for cholinergic pathways and one for autism-associated genes. Gene ontological enrichment analysis was conducted within the Pathway Studio environment ∗,” identifying any word beginning with cholin- , was used to identify cholinergic-relevant gene sets encompassing a total of 345 genes, which constituted the cholinergic activity gene set for this ontological enrichment analysis. An autism gene set containing 47 genes from a recent cross-trait genome-wide meta-analysis —one of the most widely used repositories of thoroughly annotated gene sets, used in research on both neurological conditions and on cholinergic pathways, specifically—was chosen as a precurated resource for gene sets connected with cholinergic activity . A key wanalysis  was usedanalysis .p-values (p < 0.01). A shift in the magnitude of p-values, nearest the lowest 100 p-values, served as the final determinant of cut-off for each respective gene sets’ ontology lists. The overlaps were identified using the Venn diagram web tool, Venny 2.1  per tool sorting specifications. The main two tool specifications used in this exploratory analysis were the Pathway Studio options to view first degree connections (known in Pathway Studio as Direct Interactions) and to view the shortest path between entities within the literature (known in Pathway Studio as Shortest Path). Both of these tool specifications were the primary means of building a broader understanding and figure illustration of literature-supported cholinergic networks related to both genes within the autism gene set and to theoretical cholinergic signal disruption related to dietary choline deficiency.Shared ontologies and associated pathways were rendered graphically using Pathway Studio’s NLP-driven platform. Additionally, certain elements of interest from the ontological overlap and the intersection between pathways involving genes from these sets were inserted.Graphical models were developed to examine specific pathways or networks with key autism-associated genes, and respective genetic variants which may alter choline and/or acetylcholine levels available for neuronal sensory signal transport. Each instance of a relationship identified by NLP-based word triplet identification was graded by Pathway Studio in terms of confidence levels of 1–3, with a maximum confidence level of 3 being defined as an entity-to-entity relationship supported by at least three peer reviewed publications reflected by the graphical rendering. For this study and related models, the confidence threshold for including elements in graphical representation was set at 3.Splice site identification algorithm Alternative Splice Site Predictor (ASSP)  was usedp < 0.05), namely, those for ion transport regulation and positive ion transport regulation  to ionic signal transduction down the axon is a core aspect of sensory signal pathway functionality .ex post facto. The enzyme acetylcholinesterase functions by cleaving acetylcholine in the synapse, including either acetylcholine recently made available for signal transduction, or acetylcholine released from membrane-bound acetylcholinergic receptors in the signal recipient neuron  and preserves acetylcholine in the synaptic cleft from destruction. The signal propagated through the GABAB1 receptor is, therefore, hypothesized to modulate an availability of acetylcholinesterase within the cleft. In other words, GABA-ergic suppression of ACHE upregulation may lead to decrease of synaptic acetylcholine degradation, leaving more acetylcholine available for cholinergic signal transmission. This cascade’s impact upon synaptic acetylcholine levels is depicted in B1 binding activity upon cholinergic sensory signal modulation is depicted in The regulatory role of GABA-ergic binding in acetylcholinergic activity is nuanced. Typically, GABA is released from the signal-transmitting neuron synchronously with acetylcholine through separate GABA-specific vesicles, which serve as an immediate signal modulator. The subsequent cascade that arises from GABA binding to the GABAOther modulators of acetylcholinergic signal transmission include SK2 ion channels, which counter the membrane depolarization in the signal recipient neuron . These tex post facto modulator of cholinergic activity. The autism-associated KCNN2 variant may either change the level of the baseline SK2 channel expression or lead to the alteration of the splicing, and the shift of the open reading frame. In either case, sensory signal stands to be impacted. These hypothetical impacts of a KCNN2 genetic variant upon cholinergic sensory signal can be seen in In part, the magnitude of the sensory signal depends upon SK2 channels’ ability to effectively counter membrane depolarization in the wake of a cholinergic signal, thus, serving as an GABBR1-related pathways can be seen in KCNN2-related pathways can be seen in The final modeling stage sought to evaluate and depict these hypothesized sensory signaling outcomes in light of a deficiency sufficient to reduce available acetylcholine. The intersection of dietary choline deficiency with GABBR1 and KCNN2, both the wildtype loci and the variant loci representing each of these two genes were compared using the Alternative Splice Site Predictor (ASSP) tool. While the (rs740883) SNP-related differences in predicted splicing patterns of GABBR1 were minimal, the KCNN2 variant rs13188074 yielded a high confidence prediction of a lowered strength within the splice site between the seventh and eighth exons  . Our stuGABBR1 gene encodes for the membrane protein known as GABAB1 (or sometimes GABAB). This membrane protein, which constitutes the metabotropic class of GABA-binding membrane-bound receptors, functions at a slow, steady level, in partnership with another membrane-bound GABA-binding protein, GABAB2. Through activation of potassium ion channels that release potassium (K+) out of the neuron and inhibition of calcium ion (Ca2+) channels which permit passage of Ca2+ ions into the neuron, this set of proteins brings about membrane hyperpolarization. In this circuit, GABA–GABAB1 binding activity serves an inhibitory function, which counters the possibility of excitation of membrane depolarized neurons. This functionality helps to modulate a magnitude of incoming stimulation by inhibiting sensory signals transmitted by cholinergic membrane depolarization. Thus, activation of neuronal action potentials and a subsequent, consistent state of neuronal overexcitation are prevented , an enzyme that degrades acetylcholine in the interneuronal junction, thereby lessening the magnitude of cholinergic signal. If GABA–GABAB1 binding activity were to decrease as a result of a presence of a genetic variant in a GABBR1 gene—for example, rs740883, which is strongly associated with autism—an increase in acetylcholinesterase production would ensue after being prompted by an increase in the binding of CREB1 ACHE promoter. In this scenario, the increased supply of acetylcholinesterase would degrade acetylcholine in the interneuronal junction faster, thus suppressing cholinergic signaling to a greater extent than in the brain with wildtype GABBR1. GABBR1’s autism-associated intronic variant on GABA-ergic and cholinergic signaling.Notably, the binding of GABAe supply , 6A. MorGABBR1 variant may result in cholinergic signaling being lowered further, or even markedly impaired. Moreover, given that GABA sometimes co-transmits with acetylcholine, reduced acetylcholinergic signaling may also be tied to the temporal restriction on the GABA-driven neuronal inhibition as a secondary event. Sensory processing depends upon function of both of these neurotransmitters, so it is reasonable to extrapolate that the combination of dysregulated ion transport due to the presence of GABBR1/GABAB1 variants and dietary-driven acetylcholine deficiency may result in altered sensory processing for the individual in question.Both our modeling and limited experimental evidence suggest that insufficient dietary intake of a choline may contribute to a reduced acetylcholine supply in the brain . The comKCNN2 encodes for the membrane-bound SK2 calcium-activated potassium ion channel, which modulates the excitability of neurons and neuromuscular activity. After being activated through co-located acetylcholine nicotinic receptors, SK2 channel modulates cholinergic signals through neuronal membrane repolarization  may be compounded by the synaptic acetylcholine deficiency. Thus, dietary choline deficiency may ultimately exacerbate genetic weakness in the SK2 activity, resulting in altered sensory processing, possibly resulting in sensory over-responsiveness, under-responsiveness, or a combination of both, for a given autistic person.Because SK2 signaling depends upon the signals from co-located cholinergic nicotinic receptors, diminished or altered SK2 activity arising from the presence of autism-associated GABBR1 and KCNN2 on the function of the respective proteins. ASSP analysis hints that, in case of KCNN2, the change in a strength of a splice site may influence an amount of functional mRNA encoding for full-size KCNN2. Experimental gauging of the impact of KCNN2 variant upon sensory processing is warranted.Currently, there is a little evidence of direct impact of intronic variants in GABBR1 may lead to a smaller pool of available acetylcholine  do not meet their choline AI level recommended respective of age . A numbeThe recommended dietary consumption levels for choline are set by the Food and Nutrition Board of the National Academies as assessed by measuring serum alanine aminotransferase levels reflecting overall liver function. Whether or not the choline at AI level would be sufficient to modulate sensory processing symptoms is not known. It is notable, though, that most Americans do not achieve even this level of the choline intake . For autTo call dietary intervention for choline status “low risk” does not negate the challenges specific to supporting the increased consumption of choline in autistic children and adults. The research and development which is required to determine targeted, effective ways for elevating choline consumption and identifying personalized intake baselines sufficient for fulfilling physiological need for choline in this population is considerable. As with any autism research, investigations should purposefully include representatives and researchers with an autism diagnosis who can help to shape such interventions optimally for autistic children and adults .GABBR1 and KCNN2 genetic variants have been highlighted as such only recently, and therefore there is an opportunity to collect more definitive experimental data, which can inform the modeling of sensory processing dynamics as they relate to these variants. Studies of GABBR1 and KCNN2 in autism should examine possibilities such as structure/function differences in the autism-associated variant version of these membrane proteins, as well as measurement of GABBR1 and KCNN2 expression levels in those presenting with the respective autism-associated variants.The autism-associated GABBR1 and KCNN2 would result in transcriptomic changes, transcriptomics should be a primary focus of future research on these genetic variants’ impact. Because of the measured difference in GABAB1 protein expression in autism, examining a potential link between the autism-associated GABBR1 variant and these expression differences should be a priority. Similarly, SK2 channel expression levels in autism should be examined with respect to this SK2 channel variant. Any transcriptomic differences which would be found to be associated with these genetic variants would set the foundation for more refined examination of impact upon cholinergic signal transmission and modulation.Because of the relative likelihood that autism-associated intron variants such as those found in B1 and SK2 channel structure and/or function, there are several tools and methods available, which vary in scope and performance. For example, if it can be determined that a reading frame shift has occurred as a consequence of an altered splice site, basic computational homology modeling may be helpful. Without the context of a frame shift, if structural differences are still suspected for the variant version of either of these proteins, validated structural assessment through means of electron microscopy, NMR spectroscopy, and X-ray crystallography remains as resource-intensive possibilities. Additionally, new structure prediction AI presented by AlphaFold, exhibiting unprecedented accuracy in structural prediction, could be considered as a potential future tool . Such teams may include researchers from a broad range of fields including nutrition, neuroscience, occupational therapy, bioinformatics, and computational biology. Additionally, choline-related biomarkers such as serum alanine aminotransferase may provide useful outcome measurements, particularly in cases where nutritional assessment indicates risk of deficiency.In terms of clinical interventions, while available evidence on dietary interventions for autism remains limited, this work provides mechanistic support for further exploration of exogenous choline—through diet or supplementation—as a potential low-risk sensory processing support intervention. Future clinical interventions in humans will In connecting dietary choline intake as a mediator of acetylcholinergic pathways, especially for sensory signal ion transport in the context of two autism-associated genes, GABBR1 and KCNN2, this study is consistent with the growing evidence base concerning the role of choline in pre- and postnatal nutrition and neurodevelopment. In our opinion, clinical evaluation of choline intake interventions with respect to validated sensory processing scores is warranted in future studies whose age ranges include children, teens, and adults.Because of the physically, mentally, and logistically demanding requirements involved in collection of experimental data through clinical trials, particularly from participant individuals with autism and their families, it is incumbent upon researchers to ensure that clinical trials examining dietary status, genetic variants, and sensory processing are effective, efficient, and respectful of their participants. Advance planning and implementation for these clinical trials is therefore at a premium. Accordingly, in preparation for clinical trials, research employing computational biology, bioinformatics, and predictive modeling should also be considered as essential precursor elements in examining the intersection of dietary choline status with sensory processing in the context of autism-associated genes.The autism GWAS dataset is available as a supplementary file for the original article .AO, AB, and MS were responsible for the conception of the project and editing the final manuscript. AO carried out analyses and interpretation with input from all other authors and drafted the manuscript with feedback from all other authors. All authors contributed to the design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D “tissues” called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research. The mammary gland is structured for optimal synthesis, secretion and ejection of milk products. The secretory tissue, composed of multiple cells assembled into lobes grouped into lobules, is located upstream the nipple (human) or the udder (cattle) relative to the position of both the teat and the gland cistern (in cattle). Lobules are formed of alveoli, or acini, that represent the functional milk production units in the mammary gland. The secretory tissue is drained by a tree network of ducts for the collection of milk which, in ruminants, ends up in the cistern for storage until milking or suckling. The acinus or alveolus consists of a layer of polarized mammary epithelial cells whose apical pole surrounds the alveolar lumen containing the milk they secrete and whose basal pole interacts with contractile myoepithelial cells and the basal laminae. These are embedded within the stromal tissue that contains extracellular matrix proteins, fibroblasts, adipocytes, and lymphatic and blood vessels Figure .Figure 1The development of the female mammary gland occurs during defined physiological events related to sexual development and reproduction stages, from fetal life to lactation and beyond . During Studies of mammary cells biology are of the utmost importance to understand the organization, development and functioning of the acini, and to identify significant clues for the maintenance of mammary gland function and architecture. Cell culture is essential for exploring the processes underlying the development of this organ, such as branching morphogenesis, and the dissection of its biological functions. Our aim here is to provide an overview of the development of three-dimensional (3D) cell culture systems, including current knowledge and advances in the emerging organoid culture systems, for the study of mammary gland biology in farm animals, with a focus on cattle.It is well recognized that two-dimensional 2D) cell culture does not provide optimal conditions to reach the complex organization and the specific cellular contacts observed in the mammary gland in vivo. Indeed, once mammary epithelial cells are removed from their native tissue environment and cultured on standard plastic supports, they lose essential interactions with their microenvironment such as the extracellular matrix (ECM), the other cells of the epithelium including myoepithelial cells, or the stroma. As a direct consequence, their phenotype and morphology change and they lose polarity. For milk-secreting mammary epithelial cells, the lack of relevant polarized morphology results in non-functional differentiation D cell cu, 4. FromDuring the development of 3D cell culture systems and organoids, technological advances have been made on several fronts: the nature of the ECMs and culture supports, the co-culture of various cell types and the composition of culture media.Over the past decades, ex vivo 3D culture assays with murine and human cells have been designed to recapitulate ductal invasion and elongation, morphogenic programs of alveologenesis, as well as functional differentiation . With th® and Geltrex, helped to model cell and tissue behaviors observed in normal development such as branching morphogenesis. With in vitro technique mixing 3D scaffolds and different culture media, it has even been demonstrated that the cells reform mammary terminal ductal-like structures with well-oriented luminal and basal cells [® and collagen gels with growth factors enriched media [® appeared to be much closer to the physiological reality of the cellular microenvironment. Unfortunately, its inconsistent composition, especially the concentrations of the growth factors and other biologically active components, confers to Matrigel® a batch-to-batch variability affecting the reproducibility of experiments. Like collagen, Matrigel® is unsuitable for certain experiments. When evaluating the role of mechanical and adhesive inputs on 3D tumor cell growth, invasion, and dissemination, Matrigel® rigidity may limit epithelial growth and impair dissemination. Beck et al. in 2013 demonstrated that tumors preferentially disseminate into collagen I but not into Matrigel®. As for collagen, they successfully tested addition of synthetic polymer mixtures to define the material properties that could induce dissemination into Matrigel® [®, a gel containing both basement and non-basement membrane molecules. Due to its composition, this gel exhibits biochemical and biological properties different from those of Matrigel® and was mainly used in human cancer studies.Nearly 30 years ago, solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (tumors) was developed as a potent hydrogel for the differentiation of functional mammary epithelial cells, due to its enrichment in specific ECM proteins and factors . These hydrogels or ECM matrices, now well-known as the commercial mouse-sarcoma derived Matrigelal cells , 11. As ed media . Composeatrigel® . Some mo® matrix together with mammary fibroblasts, Koledova et al. investigated the epithelial-stromal interactions during mammary branching morphogenesis. They obtained primary mammary epithelial organoids and fibroblasts by digesting mammary tissue followed by differential centrifugation. Isolation of fibroblasts from epithelial cells was based on a subsequent selective adhesion procedure. They then co-cultured fibrospheres pre-formed in polyHEMA hydrogel and mammospheres embedded in a mixture of Matrigel® and collagen I. The purpose of these 3D co-culture methodologies is to restore native interactions between epithelial cells and their neighboring stromal cells [Advances in biomaterials and tissue engineering science have equipped the biologist with new scaffolds and techniques. The last decade has seen the emergence of innovative technologies based on the combination of 3D matrices and others methodologies, including co-culture systems or use of procedures involving sequential proliferation/differentiation media cultures. Novel 3D cell culture systems incorporating co-culture with stromal cells such as endothelial or fibroblastic cells were developed. Using a 3D fibroblast–epithelium co-culture model in which primary mammary organoids are embedded within Matrigelal cells .More recently, digital design technology was adapted to construct mammary tissue using 3D bioprinting engineering to control the formation of organoids through the “self-assembly” of mammary epithelial cells . This inThe last few years have seen the organ-on-chip technology oncoming, combining bioengineering and microfluidics to mimic the microstructure, the dynamic mechanical properties and the biochemical functionalities of living organs. This technology consists of designing a bionic cellular system on a microfluidic chip possessing the characteristics of the microfluidic technology at a level of miniaturization. It takes the advantage of controlling precisely multiple parameters such as chemical concentration gradients and fluid flows. Mainly used for studies in humans, numerous tissue models including intestine, liver, lung, tumors and muscle have been developed. To our knowledge, however, only mammary tumor cells such as ductal carcinoma cells were studied in this way , 18. NevIn parallel, many efforts have been made to develop medium conditions leading to optimal differentiation of mammary cells and to achieve milk proteins and lipids synthesis. In 1988, Aggeler et al. described a 3D culture procedure including lactogenic hormones  able to induce caseins synthesis from primary mouse epithelial cells embedded in floating collagen gels . LactogeCompared to murine and human studies, the literature on 3D cell culture systems is scarce for other species, notably farm animals. Obviously, the rodent mammary gland is the most widely studied but both its anatomy and physiology are far from being fully representative of those of the ruminant mammary gland. Moreover, the scientific challenges concerning ruminants substantially differ from those for the murine and human species. Indeed, in these cases, the main interest focuses on milk secretion processes and its upstream counterpart, the development of the mammary gland. In cattle, the investigations rather concern two main science fronts: the exploration of the phases of development of the mammary gland during mammogenesis and the reproduction of the organization and functionality of the mammary cells, both to study the entire lactation cycle, including dry off, and its regulations.Three-D cell culture systems represent pertinent tools for the ex vivo modeling of mammary morphogenesis and organogenesis studies in cattle. Following the development of 3D cell culture systems made from murine and human tissue cells, including organoids culture, these methodologies were later adapted for bovine and other livestock animals. These studies were carried out using bovine primary cells obtained from enzymatically digested mammary gland tissue. A method to isolate lobules of acini from the bovine mammary gland and to further grow acini-derived cells in collagen and floating collagen gels dates from 1982 . In this®, in contrast to cells grown on fibronectin or laminin substrata that showed some TER. On the other hand, αS1-casein expression was only detected in cells grown in Matrigel® and fibronectin. However, it would be relevant to look for the other caseins to ensure their lack of expression. Even so, this suggests that in 3D culture, the intrinsic properties of the ECM proteins highly affect the cell differentiation status and the cell-to-cell organization. To date, these approaches were widely used to design mechanistic studies for the analysis of the proliferative and morphogenic potential of bovine primary cells [Many researchers have questioned the influence of the ECM protein components  on mammary cell morphology and functioning. With this aim, the impact of the components of the ECM on the maintenance of cell differentiation was compared in a study using mammary epithelial cells isolated from lactating cows . Both thry cells  and to ery cells .®. These mammospheres were composed of polarized epithelial cells remaining in direct contact with basement membrane components by recreating the cell-to-cell junction using the standard tight junction proteins zo-1 and e-cadherin, as shown by confocal microscopy. Differential transcriptomic profile analysis between the mammospheres and standard monolayer culture of BME-UV1 allowed for the identification of the genes involved in the regulation of mammary gland epithelial cell polarization and function [® and Ultra low attachment culture support, vs standard 2D cell culture, on both the cell phenotypes and morphologies for the two most popular bovine mammary epithelial cell lines, namely MAC-T and BME-UV1 cells. The basal or luminal-like profile of each cell line was characterized and we found that their phenotypes clearly influenced the performances of these cells in 2D or 3D culture. Of note, these experiments provided new information on the lineage of these cell lines and their suitability for 3D culture [In parallel to bovine primary cells, 3D cell culture systems development has also been pursued with bovine mammary cell lines. Kozlowski et al. demonstrated that BME-UV1, a well-known bovine mammary epithelial cell line, formed polarized acinar structures named mammospheres within 16 days when grown with Matrigelfunction . Our gro culture .Figure 2®. Their 3D cell culture system provided appropriate conditions for polarization of the cells as evidenced by the increased expression of the cell-to-cell junction proteins β-catenin and e-cadherin. Interestingly, however, beyond 30 passages of culture, cells embedded in Matrigel® lose their ability to express caseins while maintaining their junctional capacity [® gel were found to express genes involved in milk protein (caseins) and fat biosynthesis but not those for lactose biosynthesis [® gels, revealed that lactoferrin synthesis, a protein expressed during mammary development, differed with the type of culture substrate considered, the protein accumulating intracellularly when cells were grown within Matrigel® [Validating a 3D cell culture system in which cells differentiate to synthesize and secrete the constituents of milk, i.e., fully mimicking the secretory activity of acini, remains a challenge. Zhan et al. showed that primary mammary epithelial cells they immortalized from the mammary gland of lactating Chinese cows expressed three of the four caseins  when cultured within Matrigelcapacity . On the ynthesis . An elegatrigel® . Howeveratrigel® . Similaratrigel® . When gratrigel® .These various studies merely showed that the synthesis of milk constituents requires the polarization of the cells, accompanied by the establishment of cell-to-cell junctions. This was only found when mammary epithelial cells were grown within ECMs. However, the measurement of the activity of secretion due to mammary epithelial cells should not be limited to the expression of certain caseins. Other constituents  are as essential. However, efficiency and maintenance of active secretion of milk constituents by mammary epithelial cells of farm animals in 3D culture systems remains to be demonstrated. In conclusion, 3D cell culture system is clearly an important approach for the ex vivo modeling of tissue morphogenesis, organogenesis and biological functioning but, to date, the reconstitution of a fully functional miniature model of the mammary gland has not been achieved for domestic species.To date, very few studies have been conducted with organoids made from mammary gland cells of farm animals due to the delay in the development of such 3D cell culture models. Most of the work, however, was dedicated to the development of organoids from ruminants. Nevertheless, the development of species-specific in vitro models mimicking the detailed mammary gland physiology and function remains an essential alternative avenue for the study of physiological, biochemical, and immunologic functions of cattle mammary cells without resorting to animal experiments with their inherent ethical limits.®, the steroid hormones estradiol and progesterone were not only found to synergically accelerate the development of bovine mammary acini, but also to regulate the genes involved in the induction of the autophagy process occurring in the cells located at the center of the developing spheroids [An advantage of organoid culture models is that the effect of biomolecules known, or tested, to intervene in key cellular functions can be investigated in vitro at a cellular mechanistic level. For instance, using 3D cell culture of the bovine mammary cell line BME-UV1 embedded in Matrigelpheroids . Using tpheroids . In the pheroids . These s®. This 3D cell culture system allowed them to study the local cellular mechanisms regulating the transfer of pathogens  into milk [Organoid cultures might also help understanding the molecular events occurring during infections targeting the mammary tissue and their dynamic, e.g., during mastitis which is one of the main health problems in the dairy sector. Today, research focuses on better understanding bacterial or viral infection processes that occur in the mammary gland, and the associated immune response, in order to find alternative drugs and treatments to fight the frequent mastitis. Molecules with antimicrobial, anti-inflammatory, immunostimulatory, and antioxidant properties, notably those found in plant, are now considered to prevent or to treat mastitis. One of the research axes concerns the effects of the contact of bacteria with the somatic and epithelial cells of the mammary gland, an interaction which would induce an innate immune response in vivo. This issue can clearly be investigated using organoid culture. It should be mentioned that the main limitation of these cell culture models resides in the absence of immune cells within the system to study the inflammatory response in response to bacterial exposure in situ. To overcome this bottleneck, Le Jan et al. in 2005 developed an organoid culture model reconstructing the caprine mammary tissue, by adding blood immune cells isolated from peripheral blood mononuclear cells of goats to primary mammary cells grown within Matrigelnto milk . Combini® [® vs 3D culture supports [® or standard 2D conditions. Obviously, these methodologies present the huge advantage of using a non-invasive source of cellular materials, the mammary epithelial cells exfoliated into milk, thus preserving animal welfare. One disadvantage of this method resides in the fact that a small part of the cells recovered in milk are epithelial cells that have detached from their tissue microenvironment and stood for an undefined time in milk, probably changing their phenotype.Another approach to investigate the molecular processes underlying diseases of the bovine mammary gland has been developed by Hillreiner et al. in 2017 whose team sought to establish a 3D cell culture model using primary bovine mammary epithelial cells collected from fresh milk and grown within Matrigel® . The suisupports . Cells i® [It has to be mentioned that some toxicological tests were carried out using 3D cell culture or organoids making it possible to measure the impact of toxins on mammary tissue. Intoxication by the mycotoxin Aflatoxin B1 is known to be a public health issue and to induce cancer. The cytotoxicity of this mycotoxin and its deleterious effects on bovine mammary epithelial cells during long-term exposure was demonstrated using bovine primary mammary epithelial cells grown in a 3D cell culture system using Matrigel® . ClearlyIn ruminants raised to produce milk, understanding the development of the secretory tissue is particularly relevant to apprehend and modulate tissue plasticity in adults during their reproductive cycles. Despite exponentially growing studies using 3D cell culture models in farm animals over the past decade, cattle mammary gland organoids culture models are still in their infancy. Many efforts have been made to adapt the protocols developed with the murine and human species but in vitro research on farm species is still lagging. Major agronomic questions can be tackled with organoid culture, such as testing key biomolecules  or exploring the dynamics of mammary tissue, and that without resorting to animals. By facing the new innovative challenges to develop 3D cell culture systems, the organoid culture is expected to significantly support the agronomic research on farm animals.