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This article studies the impact caused by the success and dissemination of Broussais' theories on the use of leeches as a medical supply on Spanish–French trade relations, as well as its consequences for the Spanish market between 1821 and the 1860s. Analysing the documents produced by the different public administrations, together with newspaper and archival sources in both Spain and France and the literature and legislation of that period, allows us to understand the evolution of this trade and the heavy impact it had on the autochthonous population of this animal resource. The article reveals how, at the beginning of the 1820s, leeches became an important medical supply and how the demand for them increased significantly. This gave rise to a trade relation between Spain and France that led to the overexploitation of the resource, the issuing of regulations on the matter, and the search for technological solutions to increase the production of leeches. [ABSTRACT FROM AUTHOR]

Abstract Background Medicinal leech therapy, otherwise known as hirudotherapy or leeching, has been utilized for numerous pathologies over centuries. The technique resurfaced in modern medicine for its beneficial effects in microsurgical complications, particularly venous congestion. Recently, several cases have documented the utilization of medicinal leech therapy for the management of urologic disease states and surgical complications. It is important to understand the past, present, and future possibilities for hirudotherapy in urology. However, a systematic review of medical leech therapy in this context has yet to be conducted. Accordingly, and with an emphasis on salient clinical details, this study aims to systematically review the cases of hirudotherapy applied to urological conditions. Main body of the abstract A systematic review of cases in MEDLINE/PubMed was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Of 49 search results, 11 articles detailing 13 unique cases of medicinal leech therapy used in the urological context were included. Hirudotherapy was conducted in 10 of 13 cases (76.9%) for the management of congested blood occurring in five cases post-penile replantation, two cases of postoperative scrotal hematoma, two cases of venous congestion of the glans post-neonatal bladder exstrophy repair, and one case of refractory priapism. Leeching in the remaining three cases was for the management of penoscrotal edema in patients with hormone refractory prostate carcinoma treated with radical radiation therapy. All 13 cases (100%) reported clinical improvement from hirudotherapy. Venous congestion/hematoma cases noted decreased displaced blood volume. The patient experiencing priapism reported decreased pain despite the persistence of cavernosal swelling. The three cases of penoscrotal edema reported significant decreases in swelling due to serous fluid drainage from leech puncture sites. No complications or side effects were reported in the nine adult patients. The four postoperative neonatal patients all required blood product throughout treatment. Short conclusion The systematic review of the cases demonstrates that medicinal leech therapy is a favorable treatment option for individuals with varied urological pathologies and that hirudotherapy may improve post-surgical outcomes in the urological setting. Hirudotherapy should be considered as a viable treatment modality in specific venostatic urological conditions.

Copyright of Journal of Traditional Medical Complementary Therapies is the property of Turkiye Klinikleri and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Ранее с помощью протеолитического фермента — проназы, разрушающей глиальные клетки, на основе нервного ганглия медицинской пиявки создана нервная структура, состоящая исключительно из нейронов, соединенных электрическими синапсами — своеобразная безглиальная нервная система. Было показано, что при отсутствии глии на отростках нейронов в ганглии происходит формирование электрических синапсов (ЭС). Исследование импульсной активности одного из нейронов в ганглии — нейрона Ретциуса (НР) показало, что вместо одиночного спонтанного спайка, характерного для нейрона в норме, НР генерирует пачку импульсов реверберационного типа. Каждая пачка состоит из 3–7 импульсов, количество которых характерно для каждого нейрона. Длительность первого импульса в пачке равна длительности спайка нейрона в контроле (в среднем 6 мс), а длительность всех последующих в три раза меньше (в среднем 2 мс). Статистический анализ длительности импульсов позволил предположить, что первый импульс в пачке — собственный спонтанный спайк нейрона, а последующие импульсы — это производные от первого, сформированные электрическими синапсами на его отростках и возвратившиеся к нейрону по его нервному волокну антидромно. Путем графического моделирования проанализирован механизм формирования пачечной активности нейрона. Показано, как при преодолении одним импульсом цепочки электрических синапсов на отростках нейрон может генерировать пачечную активность реверберационного типа.

Copyright of Journal of Traditional Medical Complementary Therapies is the property of Turkiye Klinikleri and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This article aimed to determine the experts' opinion on the use of hirudotherapy in cosmetology, to supplement knowledge, and to identify factors that, in the opinion of experts, affect the effectiveness and frequency of use of this method in eliminating skin problems. The study was conducted using the expert interview method. The research tool was a categorized expert interview questionnaire using the CAWI (Computer Assisted Web Interview) technique. Hirudotherapy is one of the oldest healing methods. The mechanism of action is based on the therapeutic properties of bioactive substances isolated from leech saliva. It has been shown to have a wide range of applications in the treatment of numerous diseases in various fields of medicine, including dermatology and cosmetology. Despite its therapeutic properties and effectiveness, hirudotherapy is a rarely used tool in cosmetology. It has been found effective in the treatment of many dermatoses. The most spectacular effects have been observed in the treatment of rosacea, local inflammations, lipomas, skin scars, hard to heal wounds and contusions. [ABSTRACT FROM AUTHOR]

Simple Summary: Morganella morganii is a motile, non-spore-forming, rod-shaped facultative an-aerobic gram-negative bacterium found in the intestines of people, the oral cavity of animals, and the environment. Reptiles, guinea pigs, rabbits, jaguars, elephant seals, broiler chickens, piglets, and dolphins have all been documented to have M. morganii infection. Medicinal leeches are used in surgical and non-surgical manners. Treatment of long-term and chronic pain syndrome induced by degenerative diseases in a non-surgical method. For the first time in Iran, our investigation discovered M. morganii-infected Hirudo medicinalis. Infection with M. morganii caused a significant death and morbidity rate (70%) and severe clinical abnormalities. Medicinal leeches (Hirudo medicinalis) are used in surgical and non-surgical manners. Morganella morganii is an opportunistic and zoonotic pathogenic bacterium causing serious clinical complications. In this study, we isolated, discovered and characterized M. morganii-infected H. medicinalis. We detected and identified M. morganii in all inflamed and swollen Hirudo medicinalis samples. The 16S rRNA sequence of the isolates confirmed all strains of M. morganii. All strains were sensitive to Ceftriaxone, Ceftiofur, Danofloxacin, Ciprofloxacin, Enrofloxacin, Oxytetracycline, and Meropenem and were resistant to Erythromycin, Amoxicillin, Ampicillin, Cefazolin, Colistin, Penicillin G, and Lincomycin. This pathogenic bacterium is a zoonotic pathogen, and monitoring the prevalence rate of this bacteria is strongly necessary for leeches used in human medical treatment and care. Finally, all infected leeches were treated successfully in this case report study. [ABSTRACT FROM AUTHOR]

Abstract Background Leeches are classic annelids that have a huge diversity and are closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients. Results In this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome size. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20 and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine patterns. Finally, we systematically explored and compared the active substances in the genomes of three leech species. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules. Conclusions This study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.

Objective: Prevention and treatment of venous crisis after reconstructive microsurgery is complicated and every clinician has concern about it. We present our clinical experience with blood-sucking leech therapy for replantation of severed fingers. Methods: Blood-sucking leech therapy was prescribed for venous crisis due to various causes from July 2019-2020 in 8 patients whose distal fingers were severed. Results: After clinical application, their venous congestion was relieved, all the affected fingers survived, and the curative effect was satisfactory. Conclusion: In venous crisis after the replantation of severed fingers, blood-sucking leech therapy has a significant effect which can successfully prevent finger death caused by congestion, reduce the rate of necrosis, and improve survival rate. It is worthy of applying in clinical practice.

Abstract Background Testicular torsion/detorsion triggers tissue ischemia/reperfusion, leading to reactive oxygen species overgeneration and apoptosis. The saliva of leeches is full of anti-inflammatory, anticoagulants, antioxidants, and antimicrobial agents. Therefore, this study aimed to assess the protective mechanism of leech therapy on testicular ischemia/reperfusion damage. Methods 18 adult male rats were randomly divided into three groups: 1-Sham-operated group (SO). 2-Torsion/detorsion (T.D) group: two hours of testicular torsion with two hours of testicular detorsion was performed. 3-Torsion/detorsion + Leech therapy (TDL) group. Sperm parameters (motility, vitality, morphology, and concentration), oxidative stress biomarkers (MDA, CAT, GPx, and TAC), histopathological factors (Mean seminiferous tubular diameter, Germinal epithelial cell thickness, Testicular capsule thickness, Johnson’s score, and Cosentino’s score), and immunohistochemical markers for apoptosis detection (Bax, Bcl-2, and Caspase-3) were measured. Results There was a significant difference for all sperm parameters in the T. D group compared to the sham group. Leech therapy significantly increased progressive motility and normal morphology and reduced non-progressive motility. In the TDL group, MDA concentration significantly reduced, and levels of GPx, TAC, and CAT remarkably increased. All evaluated histopathological parameters in the TDL group significantly increased compared to the T. D group except for the testicular capsule thickness. T. D notably increased the expression of Bax and Caspase-3, while the treatment group slowed the rate of apoptosis compared to the control group. Bcl-2 expression in the T. D group was significantly lower than that in the sham group. Leech therapy increased the Bcl-2 expression. Conclusion Leech therapy attenuates damages to testicular tissue following torsion/detorsion due to its antioxidant, anti-inflammatory, and anti-apoptotic effects. Hence, it can be considered as an effective remedy for testicular ischemia/reperfusion. Graphical abstract

Mass spectrometry-based protein analysis has become an important methodology for proteogenomic mapping by providing evidence for the existence of proteins predicted at the genomic level. However, screening and identification of proteins directly on tissue samples, where histological information is preserved, remain challenging. Here we demonstrate that the ambient ionization source, nanospray desorption electrospray ionization (nanoDESI), interfaced with light microscopy allows for protein profiling directly on animal tissues at the microscopic scale. Peptide fragments for mass spectrometry analysis were obtained directly on ganglia of the medicinal leech (Hirudo medicinalis) without in-gel digestion. We found that a hypothetical protein, which is predicted by the leech genome, is highly expressed on the specialized neural cells that are uniquely found in adult sex segmental ganglia. Via this top-down analysis, a post-translational modification (PTM) of tyrosine sulfation to this neuropeptide was resolved. This three-in-one platform, including mass spectrometry, microscopy, and genome mining, provides an effective way for mappings of proteomes under the lens of a light microscope.

Microscale connectomics involves the large-scale acquisition of high-resolution serial electron micrographs from which neuronal arbors can be reconstructed and synapses can be detected. In addition to connectivity information, these data sets are also rich with structural information, including vesicle types, number of postsynaptic partners at a given presynaptic site, and spatial distribution of synaptic inputs and outputs. This study uses serial block-face scanning electron microscopy (EM) to collect two volumes of serial EM data from ganglia of the medicinal leech. For the first volume, we sampled a small fraction of the neuropil belonging to an adult ganglion. From this data set we measured the proportion of arbors that contained vesicles and the types of vesicles contained and developed criteria to identify synapses and to measure the number of apparent postsynaptic partners in apposition to presynaptic boutons. For the second data set, we sampled an entire juvenile ganglion, which included the somata and arbors of all the neurons. We used this data set to placd our findings from mature tissue in the context of fully reconstructed arbors and to explore the spatial distribution of synaptic inputs and outputs on these arbors. We observed that some neurons segregated their arbors into input only and mixed input/output zones, that other neurons contained exclusively mixed input/output zones, and that still others contained only input zones. These results provide the groundwork for future behavioral studies. J. Comp. Neurol. 524:3677-3695, 2016. © 2016 Wiley Periodicals, Inc.

Background : Leeches are classic annelids that have a huge diversity and are closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients. Results : In this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome size. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20 and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine patterns. Finally, we systematically explored and compared the active substances in the genomes of three leech species. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules. Conclusions : This study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches. [ABSTRACT FROM AUTHOR]

Purpose To describe challenges in the management of prophylaxis against infections for patients receiving medicinal leech therapy given changes in antimicrobial resistance patterns in the normal flora of leeches. Summary This article presents a patient case of reconstructive surgery complicated by infection associated with the use of medicinal leeches, as well as a discussion of prophylaxis in medicinal leech therapy, focusing on considerations for choosing a prophylactic agent. Conclusion Our case report highlights resistance changes in Aeromonas isolates associated with medicinal leeches and the potential for complications if isolates resistant to chosen prophylactic agents arise. When administering antimicrobial prophylaxis in patients receiving medicinal leech therapy, clinicians should be familiar with the susceptibilities of Aeromonas species but also conscious of evolving antimicrobial resistance given the extent of the consequences of infected surgical grafts. [ABSTRACT FROM AUTHOR]

Invertebrate animal models show simple behaviors supported by neural circuits easily accessible for experimentation and yet complex enough to provide necessary information on the cellular and molecular mechanisms that govern the vertebrate nervous system's function. The mechanisms underlying simple forms of learning have been extensively studied in the marine gastropod Aplysia californica, in which elementary non-associative learning of the behavioral habituation and sensitization type has been studied using the gill withdrawal reflex. A strong stimulus applied to the neck or tail improves the reflex response through heterosynaptic facilitation. The neurotransmitter serotonin is involved in both behavioral sensitization and dishabituation by acting through the second messenger cyclic adenosine monophosphate, protein kinase A, the phosphorylation of a K+ channel, causing its closure. This broadens the action potential profile, increases the influx of Ca2+ through voltage-gated Ca2+ channels, and enhances the neurotransmitter glutamate's release. Short-term memory is based on covalent modifications of pre-existing proteins, while long-term memory requires gene transcription, protein translation and growth of new synapses. Another simple invertebrate model is the leech Hirudo medicinalis. In nearly-intact preparations, the repetitive application of light electrical stimuli at the level of the caudal portion of the body wall can induce the habituation of swimming induction. At the same time, the stroke on the dorsal skin generates behavioral sensitization or dishabituation. Knowledge of the molecular mechanisms of activity-dependent forms of synaptic plasticity provides a basis for understanding the mechanisms underlying learning, memory, other forms of brain plasticity, and pathological conditions and suggests potential therapeutic interventions.

Blood sampling is a challenging procedure in many captive animals. Although manual restraint or anesthesia are usually possible, they entail intense stress and a high risk of injuries or organ failure. Blood sampling using medicinal leeches (Hirudo medicinalis) represents a promising non-invasive alternative to venipuncture; however, leech blood meal was to date used only for qualitative analyses such as genetic or serological screenings. Hence, the aim of this study was to evaluate the suitability of the leech blood sampling method for quantification of hematological and biochemical parameters. Medicinal leeches were manually applied on 67 zoo animals of eleven species, and control blood samples were obtained by venipuncture of the jugular vein. The leeches drew up to 20 ml of blood in 20 to 55 min. Although most hematological and biochemical parameters were significantly altered in leech-derived samples, their values showed strong (r = 0.62–0.79; 10/24 parameters) to very strong (r > 0.8; 13/24 parameters) correlations with venipuncture in all blood parameters, except for sodium (r = 0.39). As the parameter alterations and correlations were similar among species, simple cross-species regression formulas were sufficient to correct the alterations, thereby ensuring good repeatability between leeches and venipuncture in most parameters. Our data thus suggest that medicinal leeches can be used as a reliable non-invasive and stress-reducing alternative to standard venipuncture, even for quantitative assays. This opens new opportunities for a significant improvement to animal welfare in zoological gardens, conservation programmes, and ecophysiological research, where quantification of blood parameters is often needed.

This article aimed to determine the experts’ opinion on the use of hirudotherapy in cosmetology, to supplement knowledge, and to identify factors that, in the opinion of experts, affect the effectiveness and frequency of use of this method in eliminating skin problems. The study was conducted using the expert interview method. The research tool was a categorized expert interview questionnaire using the CAWI (Computer Assisted Web Interview) technique. Hirudotherapy is one of the oldest healing methods. The mechanism of action is based on the therapeutic properties of bioactive substances isolated from leech saliva. It has been shown to have a wide range of applications in the treatment of numerous diseases in various fields of medicine, including dermatology and cosmetology. Despite its therapeutic properties and effectiveness, hirudotherapy is a rarely used tool in cosmetology. It has been found effective in the treatment of many dermatoses. The most spectacular effects have been observed in the treatment of rosacea, local inflammations, lipomas, skin scars, hard to heal wounds and contusions.

Objective: Medicinal leeches ( Hirudo spp.) have been used for therapeutic purposes in humans since ancient times. Because of their growth conditions, leeches carry certain bacteria and endosymbionts (e.g., Aeromonas spp). In both leech farms and hirudotherapy clinics, there are no reliable antiseptics that can be used with leeches. This study aimed to determine whether methylene blue (MB) is a safe antiseptic for medicinal leeches and assess its safe usage., Methods: This study evaluated the efficacy of MB by determining lethal concentrations (LC), effective concentrations (EC), and lethal times (LT) for the medicinal leech Hirudo verbena Carena, 1820. A total of 570 H. verbana specimens obtained from a local farm were used in this study. Eighteen different concentrations of MB (between 1 ppm and 512 ppm) were tested., Results: The LC 50 and EC 50 values for H. verbana were determined to be 60.381 (53.674-66.636) ppm and 2.013 (1.789-2.221) ppm, respectively. The LT50 durations for MB concentrations of 32 and 512 ppm were calculated as 212.92 h (138.43 h-1485.78 h) and 17.82 h (8.08 h-23.90 h), respectively., Conclusion: The results show that MB concentrations between 2 and 19 ppm can be safely used as antiseptics in hirudotherapy clinics and leech farms to address bacterial concerns caused by medicinal leeches.

Medicinal leeches (Hirudo medicinalis) are used in surgical and non-surgical manners. Morganella morganii is an opportunistic and zoonotic pathogenic bacterium causing serious clinical complications. In this study, we isolated, discovered and characterized M. morganii-infected H. medicinalis. We detected and identified M. morganii in all inflamed and swollen Hirudo medicinalis samples. The 16S rRNA sequence of the isolates confirmed all strains of M. morganii. All strains were sensitive to Ceftriaxone, Ceftiofur, Danofloxacin, Ciprofloxacin, Enrofloxacin, Oxytetracycline, and Meropenem and were resistant to Erythromycin, Amoxicillin, Ampicillin, Cefazolin, Colistin, Penicillin G, and Lincomycin. This pathogenic bacterium is a zoonotic pathogen, and monitoring the prevalence rate of this bacteria is strongly necessary for leeches used in human medical treatment and care. Finally, all infected leeches were treated successfully in this case report study.

Medicinal leeches are effective in many clinical conditions. But leech associated Aeromonas infections are common and limits the benefits of leech therapy. Bacteriophages are specific bacteria hunting viruses that could be used effectively against target bacteria without side effects. There are 5 bacteriophages defined in the literature against Aeromonas spp. which their clinical use in hiridotherapy associated infections could open a way for a safer and more effective usage of medicinal leeches. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Traditional Medical Complementary Therapies is the property of Turkiye Klinikleri and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Testicular torsion/detorsion triggers tissue ischemia/reperfusion, leading to reactive oxygen species overgeneration and apoptosis. The saliva of leeches is full of anti-inflammatory, anticoagulants, antioxidants, and antimicrobial agents. Therefore, this study aimed to assess the protective mechanism of leech therapy on testicular ischemia/reperfusion damage. Methods: 18 adult male rats were randomly divided into three groups: 1-Sham-operated group (SO). 2-Torsion/detorsion (T.D) group: two hours of testicular torsion with two hours of testicular detorsion was performed. 3-Torsion/detorsion + Leech therapy (TDL) group. Sperm parameters (motility, vitality, morphology, and concentration), oxidative stress biomarkers (MDA, CAT, GPx, and TAC), histopathological factors (Mean seminiferous tubular diameter, Germinal epithelial cell thickness, Testicular capsule thickness, Johnson's score, and Cosentino's score), and immunohistochemical markers for apoptosis detection (Bax, Bcl-2, and Caspase-3) were measured. Results: There was a significant difference for all sperm parameters in the T. D group compared to the sham group. Leech therapy significantly increased progressive motility and normal morphology and reduced non-progressive motility. In the TDL group, MDA concentration significantly reduced, and levels of GPx, TAC, and CAT remarkably increased. All evaluated histopathological parameters in the TDL group significantly increased compared to the T. D group except for the testicular capsule thickness. T. D notably increased the expression of Bax and Caspase-3, while the treatment group slowed the rate of apoptosis compared to the control group. Bcl-2 expression in the T. D group was significantly lower than that in the sham group. Leech therapy increased the Bcl-2 expression. Conclusion: Leech therapy attenuates damages to testicular tissue following torsion/detorsion due to its antioxidant, anti-inflammatory, and anti-apoptotic effects. Hence, it can be considered as an effective remedy for testicular ischemia/reperfusion. [ABSTRACT FROM AUTHOR]

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified. • A novel potent anticoagulant protein was discovered in medicinal leech. • This protein is an antistasin homolog with 28 % amino acid sequence identity. • The recombinant protein was expressed in E. coli. • The protein directly inhibits thrombin and likely other coagulation cascade factors. [ABSTRACT FROM AUTHOR]

Introduction: Polycystic Ovary Syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age. Complementary medicine with a comprehensive and holistic approach in treating the diseases has become more popular in many countries. Since ancient times, leech therapy has been used to treat many diseases in Iranian traditional medicine. The present study was conducted with aim to review Iranian traditional medicine about leech therapy in polycystic ovary syndrome. Methods: In this review study, the issues related to polycystic ovary syndrome and leech therapy were studied in Iranian traditional medicine sources such as Qanun, kholasat al-hekmeh, Exir-e-aazam and Mofarrah al-gholub. Then, searching was performed in databases of PubMed, Google Scholar, Scopus, SID, and Magiran using the keywords such as “leech”, “leech therapy”, “Hirudo medicinalis”, “Hirudins” with “polycystic ovary syndrome”, “uterus”, and “oligoamenorrhea”. The review papers and clinical trial studies were searched in Persian and English languages without any time limitation, and the related materials were extracted and categorized. Results: The symptoms of PCOS such as the enlargement and stiffness of the ovaries and oligoamenorrhea were reported in ITM references entitled as “Ehtebas tams”. Leech therapy is useful in treatment of these patients by reducing the ovarian swelling, causing menstruation, eliminating waste material, as well as antioxidant and anti-inflammatory effects. Conclusion: Leech therapy can be used as a complementary treatment in patients with polycystic ovarian syndrome.

Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve-inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell-cell interactions that define the topography of neuronal arbors.

Ethnopharmacological Relevance: Leech, a classical traditional Chinese medicine for promoting blood circulation and removing blood stasis, is mainly used in the clinical treatment of cardiovascular and cerebrovascular diseases. The discovery of activity proteins or peptides in the dead and dried medicinal leech is an important task with great challenges., Aim of the Study: The aim of this study was to provide a basic proteome profile and help further discover active proteins and quality control for medicinal leeches, which would also provide insight into the research of animal medicines., Materials and Methods: Seventeen batches of dried medicinal leeches covering three species were collected from medicinal markets, which were authenticated by DNA barcoding. Then the proteome of different species leeches was profiled to reveal the significantly different proteins using label-free proteomics. The characteristic peptides were screened out based on biological pathways analysis, which were further absolutely quantified using the developed stable isotope-labeled based parallel reaction monitoring method., Results: Seventeen batches of leech materials were Whitmania pigra Whitman (WP), Whitmania laevis Whitman (WL) and Poecilobdella manillensis Lesson (PM), respectively. A total of 1,035 proteins (452 in WP, 425 in WL and 158 in PM) were identified. Among them, 90 overlapping proteins were mainly concentrated in diverse metabolic pathways and primarily localized in the cytoplasm and mitochondrial inner membrane, which mainly related to ATP binding, catalytic activity and structural molecular activity. In total of 51 uniquely expressed proteins (21 in WP, 23 in WL and 7 in PM), associated with multiple key signaling pathways, including Rap1, cGMP-PKG, PI3K-Akt, Wnt and HIF-1, etc., relevant to treating cardiovascular diseases, diabetes, cancer and even a variety of neurodegenerative diseases. Three proteins with potential bioactivities, including Neurohemerythrin, Hirudin and Eglin C, were selected as the quality makers and then quantified based on the characteristic peptides., Conclusions: This work profiled the proteome of three species of leeches, and addressed potential active proteins of the medicinal leech, which would help to provide the potential molecular mechanisms involved in disease treatment. The proteomics-based approach developed in this work is not only useful for the discovery of proteins with potential bioactivities but also helpful for the bioactivity relevant quality control of animal medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

BackgroundThe adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested.ResultsImaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth.ConclusionThe experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Venous congestion after digital replantation or revascularization threatens digit survival in the immediate postoperative period. External bloodletting, including leech therapy, provides a central role in salvage of the congested finger. Although there have been previous studies describing the initiation of leech therapy for digits experiencing venous insufficiency, few published articles and no consensus guidelines have discussed the weaning of leeches in the postoperative period. We review the current evidence behind leech therapy and offer a treatment algorithm based on available data and existing leech weaning protocols. [ABSTRACT FROM AUTHOR]

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79–94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context. [ABSTRACT FROM AUTHOR]

The hirudin‐like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech‐derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF‐Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin‐inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF‐Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin‐inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs. [ABSTRACT FROM AUTHOR]

Wound healing is a very complex process that allows organisms to survive injuries. It is strictly regulated by a number of biochemical and physical factors, mechanical forces included. Studying wound healing in space is interesting for two main reasons: (i) defining tools, procedures, and protocols to manage serious wounds and burns eventually occurring in future long-lasting space exploration missions, without the possibility of timely medical evacuation to Earth; (ii) understanding the role of gravity and mechanical factors in the healing process and scarring, thus contributing to unravelling the mechanisms underlying the switching between perfect regeneration and imperfect repair with scarring. In the study presented here, a new in vivo sutured wound healing model in the leech (Hirudo medicinalis) has been used to evaluate the effect of unloading conditions on the healing process and the effectiveness of platelet rich plasma (PRP) as a countermeasure. The results reveal that microgravity caused a healing delay and structural alterations in the repair tissue, which were prevented by PRP treatment. Moreover, investigating the effects of microgravity and PRP on an in vitro wound healing model, it was found that PRP is able to counteract the microgravity-induced impairment in fibroblast migration to the wound site. This could be one of the mechanisms underlying the effectiveness of PRP in preventing healing impairment in unloading conditions. [ABSTRACT FROM AUTHOR]

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons. [ABSTRACT FROM AUTHOR]

The rise of antibiotic resistance has necessitated the development of alternative strategies for the treatment of infectious diseases. Antimicrobial peptides (AMPs), components of the innate immune response in various organisms, are promising next-generation drugs against bacterial infections. The ability of the medicinal leech Hirudo medicinalis to store blood for months with little change has attracted interest regarding the identification of novel AMPs in this organism. In this study, we employed computational algorithms to the medicinal leech genome assembly to identify amino acid sequences encoding potential AMPs. Then, we synthesized twelve candidate AMPs identified by the algorithms, determined their secondary structures, measured minimal inhibitory concentrations against three bacterial species (Escherichia coli , Bacillus subtilis , and Chlamydia thrachomatis), and assayed cytotoxic and haemolytic activities. Eight of twelve candidate AMPs possessed antimicrobial activity, and only two of them, 3967 (FRIMRILRVLKL) and 536–1 (RWRLVCFLCRRKKV), exhibited inhibition of growth of all tested bacterial species at a minimal inhibitory concentration of 10 μmol. Thus, we evidence the utility of the developed computational algorithms for the identification of AMPs with low toxicity and haemolytic activity in the medicinal leech genome assembly. Image 1 • Twelve candidate AMPs of medicinal leech H. medicinalis were identified by the computational algorithms and synthesized. • Peptides 3967 and 536–1 inhibit the growth of Gram-positive and Gram-negative bacteria at a MIC of 10 μM. • Peptides 3967 and 536–1 exhibit low haemolytic activity with no cytotoxic effects. • Structure determination of the peptides in a membrane-mimetic environment was carried out by NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Modulating gene expression, either by transfection or by RNA-interference (RNAi), is a powerful means for studying the functions of essential genes during nervous system development and maintenance. Both approaches depend on the successful delivery of the corresponding nucleic acids into the targeted cells. Here we demonstrate the fast, accurate and highly localized biolistic delivery of plasmid-DNA and dsRNA into central neurons and muscle cells in internal layers of living leech embryos using carrier particles launched with a recently developed pneumatic capillary gun. This delivery method is used to perform RNAi to silence the expression of the axon guidance factor netrin in muscle cells and central neurons of leech embryos, and to induce ectopic expression of actin tagged with a fluorescent protein in both types of cells. We also show the biolistic transfection and staining in monolayer cultures of mammalian cells and carry out independent injection of two different lipophilic tracking dyes with a single capillary gun.

Abstract The purpose of this study was to investigate the effects of broodstock density on reproduction efficiency and survival of southern medicinal leech, Hirudo verbana which is neglected in aquaculture studies. 93 gravid leeches with a weight of 5.94 ± 1.06 g were placed in 2 L pet jars having moist peat in 5 different densities (1, 2, 4, 8 and 16 leeches L−1). The highest reproductive performance was obtained with 3.83 ± 0.29 and 3.67 ± 0.58 cocoons/broodstock, and 50.04 ± 5.63 and 45.21 ± 10.76 offspring/broodstock in 2 and 1 leeches L−1 stocking density, respectively. These groups were followed by groups of 4, 8 and 16 leeches L−1 stocking density with 2.33 ± 0.76, 1.79 ± 0.19 and 0.44 ± 0.33 cocoons/broodstock, and 34.79 ± 10.00, 23.74 ± 7.31 and 4.47 ± 4.37 offspring/broodstock, respectively. There were deaths as a result of cannibalism in groups of 8 and 16 leeches L−1, which were the densest groups. As broodstock density increased, deformities occurred in the cocoons, causing desiccation in incubation period, embryo deaths and decrease in the number of offspring. This study revealed that high broodstock density causes a significant reduction in the reproduction efficiency and cannibalism-induced mortality in production of H. verbana. In line with the findings obtained, 2 leeches L−1 broodstock density is recommended for efficient southern medicinal leech, H. verbana production, which prioritizes leech welfare in aquaculture. Highlights • High broodstock density reduces the number of cocoon and offspring produced in aquaculture of southern medicinal leech, Hirudo verbana. • As stocking density increases, deformations in cocoons that cause desiccation, embryo deaths and reduces number of offspring are occurred. • The gravidity rate significantly decreases over time in the uncrowded groups having high reproductive efficiency. • The crowded groups mostly do not terminate the gravidity despite the unfavourable conditions and continue the reproductive activity in an inefficient way. • At 8 leeches L−1 and higher densities, cannibalism-induced deaths occur. [ABSTRACT FROM AUTHOR]

Background: Migraine is an exceedingly common disorder that causes substantial pain, disability and societal burden. Unfortunately, conventional treatments are insufficient, inadequate, or associated with significant risks, such as overuse, abuse or addiction. The Unani system of Medicine has been treating migraine since centuries. Leech therapy (Irsale Alaq) has been employed successfully in severe persistent headache, mania and insomnia since decades. Aim: The purpose of our study was to evaluate the impact of post auricular leech therapy in migraine and to collect data to warrant further clinical trials. Methods: We conducted a case series on 7 patients who had failed conventional oral treatments for the migraine. After informed consent, post auricular leech therapy was done. Patients were asked to keep detailed headache calendars, documenting the number of headache days, migraine days, the intensity of headaches/ migraines and analgesic usage. We compared these parameters before and after 2 months of therapy. Results: We observed a reduction in the number of headache & migraine days and amount of painkillers used without any adverse effects. (P≤ 0.01) Improvement in quality of life was also observed. Discussion: We reviewed the literature related to the medicinal leeches (Hirudo medicinalis), used for the treatment in these cases. Leech saliva contains certain potent anesthetic, anti-inflammatory and vasodilator substances, suggesting plausible mechanisms of action in these cases. Conclusion: The preliminary findings indicate the safe and potential therapeutic role of leech therapy. So further trials should be carried out to explore the therapeutic potential of this therapy in chronic migraine. [ABSTRACT FROM AUTHOR]

Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism’s medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia. [ABSTRACT FROM AUTHOR]

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Copyright of Journal of Surgery & Medicine (JOSAM) is the property of Journal of Surgery & Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Antimicrobial peptides (AMPs) are considered a promising new class of anti-infectious agents. This study reports new antimicrobial peptides derived from the Hirudo medicinalis microbiome identified by a computational analysis method applied to the H. medicinalis metagenome. The identified AMPs possess a strong antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC range: 5.3 to 22.4 μM), including Staphylococcus haemolyticus, an opportunistic coagulase–negative pathogen. The secondary structure analysis of peptides via CD spectroscopy showed that all the AMPs except pept_352 have mostly disordered structures that do not change under different conditions. For peptide pept_352, the α–helical content increases in the membrane environment. The examination of the mechanism of action of peptides suggests that peptide pept_352 exhibits a direct membranolytic activity. Furthermore, the cytotoxicity assay demonstrated that the nontoxic peptide pept_1545 is a promising candidate for drug development. Overall, the analysis method implemented in the study may serve as an effective tool for the identification of new AMPs.