Computational approaches for evaluating morphological changes in the corneal stroma associated with decellularization.

Decellularized corneas offer a promising and sustainable source of replacement grafts, mimicking native tissue and reducing the risk of immune rejection post-transplantation. Despite great success in achieving acellular scaffolds, little consensus exists regarding the quality of the decellularized extracellular matrix. Metrics used to evaluate extracellular matrix performance are study-specific, subjective, and semi-quantitative. Thus, this work focused on developing a computational method to examine the effectiveness of corneal decellularization. We combined conventional semi-quantitative histological assessments and automated scaffold evaluations based on textual image analyses to assess decellularization efficiency. Our study highlights that it is possible to develop contemporary machine learning (ML) models based on random forests and support vector machine algorithms, which can identify regions of interest in acellularized corneal stromal tissue with relatively high accuracy. These results provide a platform for developing machine learning biosensing systems for evaluating subtle morphological changes in decellularized scaffolds, which are crucial for assessing their functionality.

Gray level co-occurrence matrix and wavelet analyses reveal discrete changes in proximal tubule cell nuclei after mild acute kidney injury.

Acute kidney injury (AKI) relates to an abrupt reduction in renal function resulting from numerous conditions. Morbidity, mortality, and treatment costs related to AKI are relatively high. This condition is strongly associated with damage to proximal tubule cells (PTCs), generating distinct patterns of transcriptional and epigenetic alterations that result in structural changes in the nuclei of this epithelium. To this date, AKI-related nuclear chromatin redistribution in PTCs is poorly understood, and it is unclear whether changes in PTC chromatin patterns can be detected using conventional microscopy during mild AKI, which can progress to more debilitating forms of injury. In recent years, gray level co-occurrence matrix (GLCM) analysis and discrete wavelet transform (DWT) have emerged as potentially valuable methods for identifying discrete structural changes in nuclear chromatin architecture that are not visible during the conventional histopathological exam. Here we present findings indicating that GLCM and DWT methods can be successfully used in nephrology to detect subtle nuclear morphological alterations associated with mild tissue injury demonstrated in rodents by inducing a mild form of AKI through ischemia-reperfusion injury. Our results show that mild ischemic AKI is associated with the reduction of local textural homogeneity of PTC nuclei quantified by GLCM and the increase of nuclear structural heterogeneity indirectly assessed with DWT energy coefficients. This rodent model allowed us to show that mild ischemic AKI is associated with the significant reduction of textural homogeneity of PTC nuclei, indirectly assessed by GLCM indicators and DWT energy coefficients.

Neuropathic pain: Mechanisms and therapeutic strategies.

The physiopathology and neurotransmission of pain are of an owe inspiring complexity. Our ability to satisfactorily suppress neuropathic or other forms of chronic pain is limited. The number of pharmacodynamically distinct and clinically available medications is low and the successes achieved modest. Pain Medicine practitioners are confronted with the ethical dichotomy imposed by Hippocrates: On one hand the mandate of primum non nocere, on the other hand, the promise of heavenly joys if successful divinum est opus sedare dolorem. We briefly summarize the concepts associated with nociceptive pain from nociceptive input (afferents from periphery), modulatory output [descending noradrenergic (NE) and serotoninergic (5-HT) fibers] to local control. The local control is comprised of the "inflammatory soup" at the site of pain origin and synaptic relay stations, with an ATP-rich environment promoting inflammation and nociception while an adenosine-rich environment having the opposite effect. Subsequently, we address the transition from nociceptor pain to neuropathic pain (independent of nociceptor activation) and the process of sensitization and pain chronification (transient pain progressing into persistent pain). Having sketched a model of pain perception and processing we attempt to identify the sites and modes of action of clinically available drugs used in chronic pain treatment, focusing on adjuvant (co-analgesic) medication.

Artificial neural networks in contemporary toxicology research.

Artificial neural networks (ANNs) have a huge potential in toxicology research. They may be used to predict toxicity of various chemical compounds or classify the compounds based on their toxic effects. Today, numerous ANN models have been developed, some of which may be used to detect and possibly explain complex chemico-biological interactions. Fully connected multilayer perceptrons may in some circumstances have high classification accuracy and discriminatory power in separating damaged from intact cells after exposure to a toxic substance. Regularized and not fully connected convolutional neural networks can detect and identify discrete changes in patterns of two-dimensional data associated with toxicity. Bayesian neural networks with weight marginalization sometimes may have better prediction performance when compared to traditional approaches. With the further development of artificial intelligence, it is expected that ANNs will in the future become important parts of various accurate and affordable biosensors for detection of various toxic substances and evaluation of their biochemical properties. In this concise review article, we discuss the recent research focused on the scientific value of ANNs in evaluation and prediction of toxicity of chemical compounds.

Analysis of Vascular Architecture and Parenchymal Damage Generated by Reduced Blood Perfusion in Decellularized Porcine Kidneys Using a Gray Level Co-occurrence Matrix.

There is no cure for kidney failure, but a bioartificial kidney may help address this global problem. Decellularization provides a promising platform to generate transplantable organs. However, maintaining a viable vasculature is a significant challenge to this technology. Even though angiography offers a valuable way to assess scaffold structure/function, subtle changes are overlooked by specialists. In recent years, various image analysis methods in radiology have been suggested to detect and identify subtle changes in tissue architecture. The aim of our research was to apply one of these methods based on a gray level co-occurrence matrix (Topalovic et al.) computational algorithm in the analysis of vascular architecture and parenchymal damage generated by hypoperfusion in decellularized porcine. Perfusion decellularization of the whole porcine kidneys was performed using previously established protocols. We analyzed and compared angiograms of kidneys subjected to pathophysiological arterial perfusion of whole blood. For regions of interest Santos et al. covering kidney medulla and the main elements of the vascular network, five major GLCM features were calculated: angular second moment as an indicator of textural uniformity, inverse difference moment as an indicator of textural homogeneity, GLCM contrast, GLCM correlation, and sum variance of the co-occurrence matrix. In addition to GLCM, we also performed discrete wavelet transform analysis of angiogram ROIs by calculating the respective wavelet coefficient energies using high and low-pass filtering. We report statistically significant changes in GLCM and wavelet features, including the reduction of the angular second moment and inverse difference moment, indicating a substantial rise in angiogram textural heterogeneity. Our findings suggest that the GLCM method can be successfully used as an addition to conventional fluoroscopic angiography analyses of micro/macrovascular integrity following in vitro blood perfusion to investigate scaffold integrity. This approach is the first step toward developing an automated network that can detect changes in the decellularized vasculature.

Hyperthermia and Serotonin: The Quest for a "Better Cyproheptadine".

Fine temperature control is essential in homeothermic animals. Both hyper- and hypothermia can have deleterious effects. Multiple, efficient and partly redundant mechanisms of adjusting the body temperature to the value set by the internal thermostat exist. The neural circuitry of temperature control and the neurotransmitters involved are reviewed. The GABAergic inhibitory output from the brain thermostat in the preoptic area POA to subaltern neural circuitry of temperature control (Nucleus Raphe Dorsalis and Nucleus Raphe Pallidus) is a function of the balance between the (opposite) effects mediated by the transient receptor potential receptor TRPM2 and EP3 prostaglandin receptors. Activation of TRPM2-expressing neurons in POA favors hypothermia, while inhibition has the opposite effect. Conversely, EP3 receptors induce elevation in body temperature. Activation of EP3-expressing neurons in POA results in hyperthermia, while inhibition has the opposite effect. Agonists at TRPM2 and/or antagonists at EP3 could be beneficial in hyperthermia control. Activity of the neural circuitry of temperature control is modulated by a variety of 5-HT receptors. Based on the theoretical model presented the "ideal" antidote against serotonin syndrome hyperthermia appears to be an antagonist at the 5-HT receptor subtypes 2, 4 and 6 and an agonist at the receptor subtypes 1, 3 and 7. Very broadly speaking, such a profile translates in a sympatholytic effect. While a compound with such an ideal profile is presently not available, better matches than the conventional antidote cyproheptadine (used off-label in severe serotonin syndrome cases) appear to be possible and need to be identified.

Tocilizumab and COVID-19: Timing of Administration and Efficacy.

Elevated concentrations of interleukin-6 have been demonstrated to be an important key factor in COVID-19 host immune impairment. It represents an important prognostic factor of harm associated with COVID-19 infection by stimulating a vigorous proinflammatory response, leading to the so-called "cytokine storm". Therefore, immunomodulatory interventions targeting interleukin-6 receptor antagonism have been investigated as potential treatments to counterbalance the host immune dysregulation and to support the advantageous effects of corticosteroids. Tocilizumab is a recombinant humanized monoclonal antibody that has gained much interest during the COVID-19 pandemic as an interleukin-6 receptor antagonist. Various early observational studies have reported beneficial effects of tocilizumab. Moreover, consequent randomized controlled trials have subsequently shown significant positive results about tocilizumab efficacy and safety, focusing on outcomes like mortality, risk of intensive care unit admission, and the need for mechanical ventilation, while others presented conflicting findings. In this review, we first described the pathophysiology of COVID-19 infection while highlighting the role of interleukin-6. Furthermore, we also discussed the non-conclusive evidence about tocilizumab to be used as the standard of care therapy for all patients with COVID-19 pneumonia, as well as its beneficial effects in selected patients.

Metabolic conversion of ß-pinene to ß-ionone in rats.

Exposure to or ingestion of turpentine can alter the scent of urine, conferring it a flowery, violet-like scent. Turpentine's effect on urine was initially noticed after its use either as medicine or as a preservative in winemaking. Regardless of the source of exposure, the phenomenon requires metabolic conversion of turpentine component(s) to ionone, the molecule mainly responsible for the scent of violets.The purpose of this study was to identify the presence of ionone in the urine of rats that received ß-pinene, and thus to demonstrate that the postulated conversion occurs.We treated rats intraperitoneally with normal saline (negative control), ß-ionone (positive control), low-dose ß-pinene (1/3 of LD50), and high-dose ß-pinene (1/2 of LD50). Urine samples were collected up to 72 h after administration of the compounds, followed by gas chromatography/mass spectrometry identification of the presence of ionone.ß-Ionone was found in the urine of rats exposed to both low and high doses of ß-pinene. In contrast, α-ionone appears unlikely to have been formed in rats exposed to either low or high doses of ß-pinene. ß-pinene was converted to ß-ionone, followed by partial excretion in the urine of rats. ß-Ionone is a minor metabolite of ß-pinene.

On saline infusion, clonus, molecules and forgotten scientists: Who was Dr Julius Sander (1840-1909)?

Zitterbewegungen des Fusses bei Dorsalflexion (shaking movements of the foot upon dorsal flexion) were observed independently from each other and described in the same issue of a German peer reviewed journal by Carl Westphal (1833-1890) at the Charité in Berlin and by Wilhelm Erb (1840-1921) in Heidelberg. While Westphal used the term Fussphaenomen, Erb is credited with coining the term clonus for the phenomenon. Both scientists are immortalized by various eponyms acknowledging their respective contributions to science. Little is known however about Julius Sander (1840-1909), in those days resident at Charité, who noticed the phenomenon and presented it to his superiors, Wilhelm Griesinger (1817 -1868) and Westphal. In addition to such observations, Sander made original contributions in resuscitation physiology while working with Hugo Kronecker (1839-1914). With Kronecker, Sander published observations on life saving transfusions with inorganic salt solutions in dogs "Bemerkung über lebensrettende Transfusion mit anorganischer Salzlösung bei Hunden" a very early work on isovolemic fluid resuscitation. The purpose of this communication is to highlight Sander's scientific contributions and to shed some light on his life, of which a German Lexicon stated that after 1870 no information on him can be ascertained anymore.

Lymph Nodes-On-Chip: Promising Immune Platforms for Pharmacological and Toxicological Applications.

Organs-on-chip are gaining increasing attention as promising platforms for drug screening and testing applications. However, lymph nodes-on-chip options remain limited although the lymph node is one of the main determinants of the immunotoxicity of newly developed pharmacological drugs. In this review, we describe existing biomimetic lymph nodes-on-chip, their design, and their physiological relevance to pharmacology and shed the light on future directions associated with lymph node-on-chip design and implementation in drug discovery and development.

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl.

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD01, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.

Remedia Sternutatoria over the Centuries: TRP Mediation.

Sneezing (sternutatio) is a poorly understood polysynaptic physiologic reflex phenomenon. Sneezing has exerted a strange fascination on humans throughout history, and induced sneezing was widely used by physicians for therapeutic purposes, on the assumption that sneezing eliminates noxious factors from the body, mainly from the head. The present contribution examines the various mixtures used for inducing sneezes (remedia sternutatoria) over the centuries. The majority of the constituents of the sneeze-inducing remedies are modulators of transient receptor potential (TRP) channels. The TRP channel superfamily consists of large heterogeneous groups of channels that play numerous physiological roles such as thermosensation, chemosensation, osmosensation and mechanosensation. Sneezing is associated with the activation of the wasabi receptor, (TRPA1), typical ligand is allyl isothiocyanate and the hot chili pepper receptor, (TRPV1), typical agonist is capsaicin, in the vagal sensory nerve terminals, activated by noxious stimulants.

The action of aripiprazole and brexpiprazole at the receptor level in singultus.

The hiccup (Latin singultus) is an involuntary periodic contraction of the diaphragm followed by glottic closure, which can be a rare side effect of aripiprazole. In contrast to the structurally closely related aripiprazole, brexpiprazole was not associated with this particular adverse drug reaction. Having two very similar drugs that differ in their ability to induce hiccups represents a unique opportunity to gain insight into the receptors involved in the pathophysiology of the symptom and differences in clinical effects between aripiprazole and brexpiprazole. The overlap between maneuvers used to terminate paroxysmal supraventricular tachycardia and those employed to terminate bouts of hiccups suggests that activation of efferent vagal fibers can be therapeutic in both instances. Recent work seems to support a pivotal role for serotonin receptors in such vagal activation. It is unlikely that a unique receptor-drug interaction could explain the different effects of the examined drugs on hiccup. The different effect is most likely the consequence of several smaller effects at more than one receptor. Brexpiprazole is a highly affine (potent) α2C antagonist and, therefore, also an indirect 5-HT1A agonist. In contrast, aripiprazole is a partial 5-HT1A agonist (weak antagonist) and an HT3 antagonist. Activation of 5-HT1A receptors enhances vagal activity while HT3 blockade reduces it. Vagus nerve activation is therapeutic for hiccups. A definitive answer continues to be elusive.

Brain delivery of antidotes by polymeric nanoparticles.

Accidental intoxications from environmental pollutants, as well as intentional self- and chemical warfare-related poisonings affect millions of people worldwide each year. While many toxic agents can readily enter the central nervous system (CNS), the blood-brain barrier (BBB) prevents the brain uptake of most pharmaceuticals. Consequently, poisoning antidotes usually cannot reach their site of action in the CNS in therapeutically relevant concentrations, and thus only provide effective protection to the peripheral nervous system. This limitation can be overcome by encapsulating the antidotes in nanoparticles (NP), which can enhance their CNS accumulation without damaging the integrity of the BBB. Among nanocarriers, polymer-based drug delivery systems exhibit remarkable benefits, such as bioavailability, cell uptake and tissue retention. Furthermore, due to their capacity to mask unfavorable physicochemical properties of cargo drugs, polymeric NPs were able to improve BBB transport of various pharmaceuticals. However, while polymer NP-mediated treatment of various pathological brain conditions, such as glioma and Alzheimer's disease were exhaustively studied, the application of polymeric nanocarriers for brain-targeted delivery of antidote molecules has not been adequately examined. To display its therapeutic potential, we review the state of the art of polymer NP-assisted CNS delivery of antidotes for various poisonings, including heavy metal and organophosphorus intoxications.

Use of Biodegradable, Chitosan-Based Nanoparticles in the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects more than 24 million people worldwide and represents an immense medical, social and economic burden. While a vast array of active pharmaceutical ingredients (API) is available for the prevention and possibly treatment of AD, applicability is limited by the selective nature of the blood-brain barrier (BBB) as well as by their severe peripheral side effects. A promising solution to these problems is the incorporation of anti-Alzheimer drugs in polymeric nanoparticles (NPs). However, while several polymeric NPs are nontoxic and biocompatible, many of them are not biodegradable and thus not appropriate for CNS-targeting. Among polymeric nanocarriers, chitosan-based NPs emerge as biodegradable yet stable vehicles for the delivery of CNS medications. Furthermore, due to their mucoadhesive character and intrinsic bioactivity, chitosan NPs can not only promote brain penetration of drugs via the olfactory route, but also act as anti-Alzheimer therapeutics themselves. Here we review how chitosan-based NPs could be used to address current challenges in the treatment of AD; with a specific focus on the enhancement of blood-brain barrier penetration of anti-Alzheimer drugs and on the reduction of their peripheral side effects.

The Role of Serotonin in Singultus: A Review.

The use of dopamine receptor blockers for chronic singultus treatment is based-at least partially-on circular thinking: chlorpromazine is FDA-approved for hiccups, chlorpromazine is a neuroleptic, neuroleptics are dopamine receptor blockers, and therefore hiccup is due to dopaminergic dysfunction. Chlorpromazine interacts with high affinity with a multitude of receptors and ion channels. This promiscuity is the basis for many of the therapeutic effects and adverse drug reactions of this drug. While an involvement of dopamine is certain, it is by no means clear that dopaminergic dysfunction is the hallmark of singultus. The common denominator of most remedies for transient hiccup is their ability to activate the vagus nerve. Both afferent and efferent vagal activity and the central integration of the Xth cranial nerve function are modulated, inter alia, via serotonergic mechanisms; beneficial (therapeutic) effects for hiccup are to be expected from serotonin (5-HT) receptor subtype ligands that enhance vagal activity. Taken together, it appears that the ability to increase vagus output is mainly associated with 5-HT1A, 5-HT3, and 5-HT7 agonists and with 5-HT2C antagonists. The plausibility of the serotonergic singultus hypothesis is examined against available pharmacokinetic, pharmacodynamic, and clinical data for a number of drugs.

Combined Pre- and Posttreatment of Paraoxon Exposure.

AIMS: Organophosphates (OPCs), useful agents as pesticides, also represent a serious health hazard. Standard therapy with atropine and established oxime-type enzyme reactivators is unsatisfactory. Experimental data indicate that superior therapeutic results can be obtained when reversible cholinesterase inhibitors are administered before OPC exposure. Comparing the protective efficacy of five such cholinesterase inhibitors (physostigmine, pyridostigmine, ranitidine, tacrine, or K-27), we observed best protection for the experimental oxime K-27. The present study was undertaken in order to determine if additional administration of K-27 immediately after OPC (paraoxon) exposure can improve the outcome. METHODS: Therapeutic efficacy was assessed in rats by determining the relative risk of death (RR) by Cox survival analysis over a period of 48 h. Animals that received only pretreatment and paraoxon were compared with those that had received pretreatment and paraoxon followed by K-27 immediately after paraoxon exposure. RESULTS: Best protection from paraoxon-induced mortality was observed after pretreatment with physostigmine (RR = 0.30) and K-27 (RR = 0.34). Both substances were significantly more efficacious than tacrine (RR = 0.67), ranitidine (RR = 0.72), and pyridostigmine (RR = 0.76), which were less efficacious but still significantly reduced the RR compared to the no-treatment group (paraoxon only). Additional administration of K-27 immediately after paraoxon exposure (posttreatment) did not further reduce mortality. Statistical analysis between pretreatment before paraoxon exposure alone and pretreatment plus K-27 posttreatment did not show any significant difference for any of the pretreatment regimens. CONCLUSIONS: Best outcome is achieved if physostigmine or K-27 are administered prophylactically before exposure to sublethal paraoxon dosages. Therapeutic outcome is not further improved by additional oxime therapy immediately thereafter.

Singultus, paper-bag ventilation, and hypercapnia.

Sir Louis Francis Knuthsen (1869-1957), the physician who painstakingly listed almost all treatments known for obstinate hiccough, ascribes the holding of breath method to Philip Henry Pye-Smith, FRS (1840-1914), consultant at Guy's Hospital in London. In fact, the strategy is much older and was mentioned by greats such as Francis Bacon (1561-1626), Aristoteles (384-322 BC), and Eryximachus (late-fifth century bce). Hypoventilation to reduce central nervous system excitability was used in antiquity as evidenced by Cyriacus' treatment of Artemia, the daughter of Emperor Diocletian (≈ 244-311). She was suffering from (among others) seizures that Cyriacus was apparently controlling by tightening a scarf around her neck, as depicted by Mathias Grünewald (1460-1528) on a wing of the so-called Heller Altar now on display at the Historical Museum, Frankfurt, Germany. In modern times, around 1920, inducing hypercapnia by CO2 inhalation as therapy for hiccups was suggested and tried by a number of anesthetists, such as Americans Russel Firth Sheldon (1885-1960) and Brian Collins Sword (1889-1956) in Boston; Briton Christopher Langton Hewer (1896-1986) at St. Bartholomew's Hospital in London; Austrian Karl Doppler (1887-1947) in Vienna; and the German/Polish Arthur Dzialoszynski (1893-1977) in Berlin. Although various authors assign the scientific primate to any of them, the first mention of carbon dioxide inhalation as treatment of singultus in the scientific literature is of French origin and was made by Paris pharmacist Henri Bocquillon-Limousin (1856-1917) in his 1892 Formulaire des médicaments nouveaux et des médications nouvelles.

Oximes as pretreatment before acute exposure to paraoxon.

Organophosphates, useful agents as pesticides, also represent a serious danger due to their high acute toxicity. There is indication that oximes, when administered before organophosphate exposure, can protect from these toxic effects. We have tested at equitoxic dosage (25% of LD01 ) the prophylactic efficacy of five experimental (K-48, K-53, K-74, K-75, K-203) and two established oximes (pralidoxime and obidoxime) to protect from mortality induced by the organophosphate paraoxon. Mortalities were quantified by Cox analysis and compared with those observed after pretreatment with a strong acetylcholinesterase inhibitor (10-methylacridine) and after the FDA-approved pretreatment compound pyridostigmine. All nine tested substances statistically significantly reduced paraoxon-induced mortality. Best protection was conferred by the experimental oxime K-48, reducing the relative risk of death (RR) to 0.10, which was statistically significantly superior to pyridostigmine (RR = 0.31). The other oximes reduced the RR to 0.13 (obidoxime), 0.20 (K-203), 0.21 (K-74), 0.24 (K-75) and 0.26 (pralidoxime), which were significantly more efficacious than 10-methylacridine (RR = 0.65). These data support the hypothesis that protective efficacy is not primarily due to cholinesterase inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the established pretreatment compound pyridostigmine.