Hirudo Medicinalis Research Articles

Evaluation of Mitotic Activity of Bone Marrow Under Short-Term Hypothermia

In this research, we have studied the mitotic activity of bone marrow when applying medicinal leeches and short-term hypothermia. The bone marrow mitotic indices were compared within an immediate examination of the thigh removed from the body and during a short-term hypothermia. As a result, no statistical differences were found between the mitotic levels during immediate examination and a short-term hypothermia in the intact animals and those after medicinal leech application, that evidenced to a hypothermal fixation of initial level of this index, preservation of biomembrane physiological state prior to hypotonic stress and cell morphology. The used here short-term hypothermia on bone marrow cells may be included in the multi-profile algorithm of laboratory analysis when studying a mitotic index in actively proliferating cells.

Effects of mammalian blood with different glucose levels on reproduction, growth and survival of the southern medicinal leech, Hirudo verbana Carena, 1820

Medicinal leeches are generally fed using pure mammalian blood. In the present study reproduction, growth and survival of medicinal leeches (Hirudo spp.) fed by mammalian blood with modified glucose level were investigated for the first time. Leeches were fed by cattle blood in a final glucose level of 152 mg/dL (control group; Glucose-free), 200 mg/dL (G200 group), 300 mg/dL (G300 group), 500 mg/dL (G500 group), 750 mg/dL (G750 group), 1000 mg/dL (G1000 group), 2500 mg/dL (G2500 group) and 5000 mg/dL (G5000 group) with the addition of D-Glucose Monohydrate. Greatest growth performance was determined in the G2500 group with a specific growth rate of 2.34% (final body weight: 10.37 ± 3.86 g) (P < 0.05). A quadratic increase was observed in the body weight values of the leeches depending on the glucose dose (Plinear and Pquadratic < 0.05). The greatest survival and gravidity rates were 89% and 38%, respectively, in the G750 group (P < 0.05). The increased glucose level caused a sharp decrease in the survival and gravidity rates of leeches. The glucose level did not significantly effected the cocoon and offspring productivity (P > 0.05). According to the broken line model, optimum glucose levels based on growth, survival rate and gravidity rate were 2461 mg/dL, 750.0 mg/dL and 749.9 mg/dL, respectively. The study showed that, although the optimum growth performance was obtained in the G2500 group, blood with glucose level of 750 mg/dL should be used for profitable medicinal leech culture considering survival and gravidity rates.

Hirudin alleviates acute ischemic stroke by inhibiting NLRP3 inflammasome-mediated neuroinflammation: In vivo and in vitro approaches

Acute ischemic stroke is a severe condition that a vessel supplying blood to the brain is abruptly blocked mostly due to cerebral thrombosis and embolism. There is a dearth of the effective prevention and early intervention strategies. NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation plays a crucial role in the pathophysiology of ischemic stroke. Hirudin is a secretion from the salivary glands of the leech Hirudo medicinalis and has a role in regulating inflammation. In this study, hirudin with a dose of 10–40 mg/kg was given to middle cerebral artery occlusion/reperfusion mice. Hirudin markedly constrained cerebral infarct area in a dose-dependent manner, and significantly improved locomotor disability at 40 mg/kg dose. Similar to MCC950, a selective NLRP3 inflammasome inhibitor, hirudin inhibited M1 polarization and promoted M2 polarization. It also strikingly suppressed the ischemia-induced overexpression of NLRP3 and its downstream components, caspase-1, apoptosis-associated speck-like protein (ASC), and interleukin-1β (IL-1β). Hirudin and MCC950 equivalently protected viability and death of BV-2 microglia cells against oxygen-glucose deprivation/reperfusion (OGD/R), an in vitro cell model of brain ischemia. Both agents had similar effects in normalizing the OGD/R-evoked aberrant microglial profiles and NLRP3 pathway dysregulation as observed in the mice. These results demonstrated anti-ischemic effects of hirudin and its association with the inhibition of microglial NLRP3 inflammasome-mediated neuroinflammation. Hirudin is a promising agent for the early intervention of acute ischemic stroke.

A Survey Study on Leech Storage Practices in Ayurveda Hospitals

Leech therapy is one of the popular treatment method adopted in Ayurveda hospitals. We have conducted a survey study to evaluate the method of collection; storage and maintenance of leeches. Data regarding the leech storage were collected from 31 Ayurveda hospitals, on the basis of a standardized questionnaire. All the responding hospitals practiced leech therapy, mostly with a storage setup in the minor OT. 68% procured leeches during rainy season from the leech suppliers. 42% responders used tap water and plastic jars for storage. 94% of Ayurveda hospitals did not subject the water used for storage of medicinal leeches for physiochemical and microbial parameters periodically. There is a need for breeding centers in order to supply of medicinal leeches throughout the year. There is a need for establishing national guidelines for good storage and maintenance of medicinal leeches in India.

Serotonin mediates stress-like effects on responses to non-nociceptive stimuli in the medicinal leech Hirudo verbana.

ABSTRACTNoxious stimuli can elicit stress in animals that produce a variety of adaptations including changes in responses to nociceptive and non-nociceptive sensory input. One example is stress-induced analgesia that may be mediated, in part, by the endocannabinoid system. However, endocannabinoids can also have pro-nociceptive effects. In this study, the effects of electroshock, one experimental approach for producing acute stress, were examined on responses to non-nociceptive mechanical stimuli and nociceptive thermal stimuli in the medicinal leech (Hirudo verbana). The electroshock stimuli did not alter the leeches’ responses to nociceptive stimuli, but did cause sensitization to non-nociceptive stimuli, characterized by a reduction in response threshold. These experiments were repeated with drugs that either blocked synthesis of the endocannabinoid transmitter 2-arachidonoylglycerol (2-AG) or transient receptor potential vanilloid (TRPV) channel, which is known to act as an endocannabinoid receptor. Surprisingly, neither treatment had any effect on responses following electroshock. However, the electroshock stimuli reliably increased serotonin (5-hydroxytryptamine or 5HT) levels in the H. verbana CNS. Injection of 5HT mimicked the effects of the electroshocks, sensitizing responses to non-nociceptive stimuli and having no effect on responses to nociceptive stimuli. Injections of the 5HT receptor antagonist methysergide reduced the sensitization effect to non-nociceptive stimuli after electroshock treatment. These results indicate that electroshocks enhance response to non-nociceptive stimuli but do not alter responses to nociceptive stimuli. Furthermore, while 5HT appears to play a critical role in this shock-induced sensitizing effect, the endocannabinoid system seems to have no effect.

The Effect of the Biologically Complex of a Medical Leech Active Substances on The Immunosuppressive State of Rats

The Effect of the Biologically Complex of a Medical Leech Active Substances on The Immunosuppressive State of Rats

Comprehensive Transcriptome Sequencing Analysis of Hirudinaria manillensis in Different Growth Periods.

Medical leeches are widely been used in biochemical and clinical medical studies, helping to restore blood circulation to grafted or severely injured tissue. Mostly, adult leeches are being used in the traditional pharmacopeia, but the gene expression profiling of leeches in different growth periods is not well-reported. So, in this study, we used transcriptome analysis to analyze the comparative gene expression patterns of Hirudinaria manillensis (H. manillensis) in different growth periods, including larval, young, and adult stages. We constructed 24 cDNA libraries from H. manillensis larval, young, and adult stages, and about 54,639,118 sequences were generated, 18,106 mRNA transcripts of which 958 novel mRNAs and 491 lncRNAs were also assembled as well. Furthermore, the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially upregulated genes from the larval to adult stages were enriched in pathways such as cilium, myofibril, contractile fiber, cytoskeleton proteins, dilated cardiomyopathy, adrenergic signaling in cardiomyocytes, etc. Moreover, in the adult stages, a significant increase in the expression of the Hirudin-HM (HIRM2) genes was detected. In addition, our comparative transcriptome profiling data from different growth stages of H. manillensis also identified a large number of DEGs and DElncRNAs which were tentatively found to be associated with the growth of H. manillensis; as it grew, the muscle-related gene expression increased, while the lipid metabolism and need for stimulation and nutrition-related genes decreased. Similarly, the higher expression of HIRM2 might attribute to the high expression of protein disulfide isomerase gene family (PDI) family genes in adulthood, which provides an important clue that why adult leeches rather than young leeches are widely used in clinical therapeutics and traditional Chinese medicine.

Management of Artery-Only Digit Replantation: A Systematic Review of the Literature.

Artery-only replantation may be necessary in circumstances when venous repair cannot be performed due to their size or vessel injury. Venous congestion of the replanted part is inevitable. A systematic review was performed to identify techniques for mitigating congestion and analyze the outcomes of those techniques. A comprehensive literature search was performed to identify relevant articles related to artery-only replantation. An initial query identified 1286 unique articles. A total of 55 articles were included in the final review. Included studies were categorized by decongestive technique. Data from each article included the number of patients treated, level of amputation, graft use, anticoagulation or antiplatelet therapy, replant viability, and follow-up duration. Weighted averages were determined from studies that reported five or more digits. A total of 1498 individual digital replantations were described. Very rarely did studies report artery-only replantation proximal to the distal interphalangeal joint. An overall survival rate of 78.5 percent was found irrespective of technique but was variable based on each particular study. Studies utilizing medicinal leech therapy were more likely to report use of intravenous anticoagulation, whereas surface bleeding techniques were more likely to report use of topical or local anticoagulant. Lack of a vein for anastomosis should not be regarded as a contraindication to replantation. These digits instead require a method to establish reliable drainage sufficient to allow for low resistance inflow and maintain a physiologic pressure gradient across capillary beds. The surgeon should select a decongestive technique that best suits the patient and their specific injury.

Initial Variability and Time-Dependent Changes of Neuronal Response Features Are Cell-Type-Specific.

Different cell types are commonly defined by their distinct response features. But several studies proved substantial variability between cells of the same type, suggesting rather the appraisal of response feature distributions than a limitation to “typical” responses. Moreover, there is growing evidence that time-dependent changes of response features contribute to robust and functional network output in many neuronal systems. The individually characterized Touch (T), Pressure (P), and Retzius (Rz) cells in the medicinal leech allow for a rigid analysis of response features, elucidating differences between and variability within cell types, as well as their changes over time. The initial responses of T and P cells to somatic current injection cover a wide range of spike counts, and their first spike is generated with a high temporal precision after a short latency. In contrast, all Rz cells elicit very similar low spike counts with variable, long latencies. During prolonged electrical stimulation the resting membrane potential of all three cell types hyperpolarizes. At the same time, Rz cells reduce their spiking activity as expected for a departure from the spike threshold. In contrast, both mechanoreceptor types increase their spike counts during repeated stimulation, consistent with previous findings in T cells. A control experiment reveals that neither a massive current stimulation nor the hyperpolarization of the membrane potential is necessary for the mechanoreceptors’ increase in excitability over time. These findings challenge the previously proposed involvement of slow K+-channels in the time-dependent activity changes. We also find no indication for a run-down of HCN channels over time, and a rigid statistical analysis contradicts several potential experimental confounders as the basis of the observed variability. We conclude that the time-dependent change in excitability of T and P cells could indicate a cell-type-specific shift between different spiking regimes, which also could explain the high variability in the initial responses. The underlying mechanism needs to be further investigated in more naturalistic experimental situations to disentangle the effects of varying membrane properties versus network interactions. They will show if variability in individual response features serves as flexible adaptation to behavioral contexts rather than just “randomness”.

A new species of medicinal leech in the genus Hirudo Linnaeus, 1758 (Hirudiniformes, Hirudinidae) from Tianjin City, China.

Medicinal leeches in the genus Hirudo have been utilized for therapeutic procedures for thousands of years. To date, six known species of Hirudo are widely distributed in different regions of the Eurasian continent. In this study, a new medicinal leech species Hirudotianjinensis Liu, sp. nov. is described based upon specimens collected from Tianjin City, China. The new species can be distinguished from its congeners by a combination of characters: blackish green dorsum with five continuous yellow longitudinal stripes; six sensillae on dorsal annulus a2 of segments VIII–XXV; greyish green ventrum with irregular bilateral dark brown spots; dorsum and abdomen separated by a pair of pale yellow stripes; front half atrium wrapped by white prostate; apparent albumen gland; epididymis massive in relation to ejaculatory bulb. The phylogenetic tree based upon COI implies a sister relationship to H.nipponia Whitman, 1886. A key to the known species is provided.

Utilization of Leech Therapy after Rhinoplasty.

The use of medicinal leeches in modern reconstructive surgery is well-described. Leech therapy after rhinoplasty has not been previously well-characterized. The medical records of all patients who underwent open rhinoplasty by a single surgeon over a 4-year period were reviewed. Patient demographics, including age, sex, medical comorbidities, number of previous rhinoplasty surgeries, time to utilization of leech therapy, adjunct therapies used, resolution of skin changes, and smoking status, were recorded. Operative reports were reviewed for pertinent information, including number of tip grafts used, graft materials used, and placement of septal extension grafts or "unicorn" grafts. Between April of 2016 and March of 2020, 545 patients underwent rhinoplasty performed by the senior author (P.S.N.). Of these patients, 39 (7.2 percent) underwent leech therapy postoperatively. The mean age of included patients was 47.4 years. Of the patients who required leech therapy, 34 (87.2 percent) had undergone revision rhinoplasty. The mean number of previous rhinoplasties was 3.4. The mean number of tip grafts used was 2.6. Thirty-three patients (84.6 percent) had either a traditional septal extension graft or unicorn graft placed. Nine patients (23.1 percent) were former smokers. Complete resolution of skin color changes was seen in 38 patients (97.4 percent). There were no major complications after leech therapy. Leech therapy is a useful tool for the rhinoplasty surgeon, particularly in the setting of complex revision rhinoplasty, in patients who have undergone multiple previous nasal surgical procedures, or in patients who require significant cartilage grafting to reconstruct the nasal tip or lengthen the nose. Therapeutic, IV.

The Medicinal Leech Possesses All Mechanisms of Influence for Preventing Pathogenesis of SARS-CoV-2

The Medicinal Leech Possesses All Mechanisms of Influence for Preventing Pathogenesis of SARS-CoV-2

A Taxonomic Study comparing the two types of Medicinal Leeches available in Iraq

Medical leeches are ectoparasites invertebrates which are widely used in medical and surgical treatments because it was found since the ancient time that many diseases can be cured by medicinal leeches, then it was discovered that leech saliva contains anti-inflammatory, analgesic, antibacterial, anticoagulant, and even anticancer enzymes, also they found that medicinal leeches have a great role in treating diabetic ulcers. Tow samples of leeches were collected from two regions in Iraq from September to the end of December 2020. These samples were examined a live by dissecting microscope and then they were examined after fixing them in formaldehyde 5%. The samples of leeches that were used to analyze their genetic DNA were fixed and preserved in absolute ethanol. The classification process was done through a molecular diagnosis of samples of medical leeches by using polymerase chain reaction PCR and technique tracking nitrogenous bases in the nucleic acid chain, and the molecular diagnosis depending on Cytochrome oxidase subunit 1 (CO1), and then the result of PCR of medical leech samples was sent with the primers of the resulting bands and the gene sequences were read depending on the Genetic Analyser Device 3130 supplied by Japanese Hitachi company. Then the gene sequences were matched with gene sequences documented in the National Center Biotechnology Information NCBI. The results were analyzed depending on the BLAST program through which two types of medical leeches were detected Hirudo medicinalis and Hirudo verbena.

Molecular dynamic and pharmacological studies on protein-engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis.

Hirudin variants are the most powerful thrombin inhibitors discovered to date, with a lower risk of bleeding than heparin. For anticoagulation, the C-termini of hirudin variants bind to the exocite I of thrombin. Anticoagulant effects of gene-recombinant hirudin are weaker than natural hirudin for the reason of lacking tyrosine O-sulfation at C-terminus. An integrative pharmacological study was carried out using molecular dynamic, molecular biological and in vivo and in vitro experiments to elucidate the anticoagulant effects of protein-engineered hirudins. Molecular dynamic analysis showed that modifications of the C-termini of hirudin variant 1 of Hirudo medicinalis (HV1) and hirudin variant 2 of Hirudinaria manillensis (HM2) changed the binding energy of the C-termini to human thrombin. The study indicated that Asp61 of HM2 that corresponds to sulfated Tyr63 of HV1 is critical for inhibiting thrombin activities. Further, the anticoagulant effects of HV1 and HM2 were improved when the amino acid residues adjacent to Asp61 were mutated to Asp. These improvements were prolongation of the activated partial thromboplastin time, prothrombin time and thrombin time of human blood, and decreased Ki and IC50 values. In the in vivo experiments, mutations at C-termini of HV1 and HM2 significantly changed partial thromboplastin time, prothrombin and thrombin time CONCLUSION AND IMPLICATIONS: The study indicated that the anticoagulant effects of gene-engineered HM2 are stronger than gene-engineered HV1 and HM2-E60D-I62D has the strongest effects and could be an antithrombotic with better therapeutic effects.

Activity-Dependent Modulation of Tonic GABA Currents by Endocannabinoids in Hirudo verbana.

Endocannabinoids are lipid neuromodulators that are synthesized on demand and primarily signal in a retrograde manner to elicit depression of excitatory and inhibitory synapses. Despite the considerable interest in their potential analgesic effects, there is evidence that endocannabinoids can have both pro-nociceptive and anti-nociceptive effects. The mechanisms contributing to the opposing effects of endocannabinoids in nociception need to be better understood before cannabinoid-based therapies can be effectively utilized to treat pain. Using the medicinal leech, Hirudo verbana, this work investigates whether endocannabinoids modulate tonic inhibition onto non-nociceptive afferents. In voltage clamp recordings, we analyzed changes in the tonic inhibition in pressure-sensitive (P) cells following pre-treatment with endocannabinoids, 2-arachidonoylglycerol (2-AG) or anandamide (AEA). We also tested whether high frequency stimulation (HFS) of nociceptive (N) cells could also modulate tonic inhibition. Both endocannabinoid application and N cell HFS depressed tonic inhibition in the P cell. Depression of tonic inhibition by N cell HFS was blocked by SB 366791 (a TRPV1 inhibitor). SB 366791 also prevented 2-AG-and AEA-induced depression of tonic inhibition. HFS-induced depression was not blocked by tetrahydrolipstatin (THL), which prevents 2-AG synthesis, nor AM 251 (a CB1 receptor inverse agonist). These results illustrate a novel activity-dependent modulation of tonic GABA currents that is mediated by endocannabinoid signaling and is likely to play an important role in sensitization of non-nociceptive afferent pathways.

The present state of the leech fauna (Annelida: Hirudinea) in Lithuania.

The aim of the present study is to present the composition of the leech fauna from Lithuania. Based on morphology, 12 species of leeches belonging to 10 genera and 5 families (Haemopidae, Hirudinidae, Glossiphoniidae, Erpodellidae and Piscicolidae) were identified in the country. Comparison of the data generated in current study with historical records, we extend the list of species to 21. Additional localities of Hirudo medicinalis and Erpobdella monostriata, two uncommon leeches, were established in Lithuania.

The effects of substrate porosity, mechanical substrate properties and loading conditions on the attachment performance of the Mediterranean medicinal leech (Hirudo verbana).

The ectoparasitic lifestyle of the Mediterranean medicinal leech (Hirudo verbana) requires reliable functioning of its attachment organs (i.e. anterior and posterior suction discs) on multiple habitat- and host-specific surfaces under both normal and shear stresses. In addition to some intrinsic properties of the attachment devices, however, only a few extrinsic factors (e.g. substrate roughness and porosity) have been considered in previous studies on leech suckers. Using centrifugal force experiments, we analysed the attachment performance of H. verbana under different types of loading on artificial substrates differing in porosity and their mechanical properties. Whereas the substrate porosity significantly influenced leech attachment under normal and shear loading, the different mechanical properties did not noticeably affect attachment within the considered parameter limits. Furthermore, suction was confirmed to be the primary attachment mechanism independent of the prevailing loading condition. The question of whether the suction cups of H. verbana are adapted to a specific loading condition could not be answered. In any case, our results again highlight the high functional resilience of leech suckers guaranteeing a successful ectoparasitic lifestyle.

Hirudin promotes proliferation and osteogenic differentiation of HBMSCs via activation of cyclic guanosine monophosphate (cGMP)/protein kinase-G (PKG) signaling pathway

ABSTRACT Osteoporosis is a public health problem resulting in higher susceptibility to bone fracture. Hirudin is known as a direct thrombin inhibitor, which is isolated from the salivary gland of the medicinal leech. The present study aimed to evaluate the effect of Hirudin on the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSCs). In our study, the effect of Hirudin on the proliferation of HBMSCs was evaluated with the CCK-8 and MTT assays. The capacity of osteogenic differentiation and mineralization of HBMSCs was evaluated with ALP and alizarin red staining, respectively. cGMP content was determined by ELISA. Western blotting and qRT-PCR were used to investigate the effect of Hirudin on the expression of osteoblast-specific markers, including Runx2, osterix (OSX), osteocalcin (OCN), and collagen1 (Col1). In our study, Hirudin treatment promoted cell viability. Moreover, Hirudin treatment increased ALP activity of HBMSCs and red coloration of alizarin. Interestingly, cGMP inhibitor partly reversed the effect of Hirudin on the proliferation, differentiation and mineralization of HBMSCs. In conclusion, Hirudin promoted the proliferation, differentiation and mineralization of HBMSCs via activation of cGMP signaling pathway. Hence, Hirudin contributed to bone remodeling and might represent as an effective agent for the treatment of osteoporosis.

Validation of Medicinal Leeches (Hirudo medicinalis) as a Non-invasive Blood Sampling Tool for Hematology and Biochemistry Profiling in Mammals.

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.

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients

BackgroundLeeches 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.ResultsIn 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.ConclusionsThis 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.