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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 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]

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]

Leech assemblages on the bottom substrata and artificial plates deployed in various types of waterbodies located in an urbanized area (Olsztyn, Northeastern Poland) were examined to identify environmental factors responsible for their distribution. The leech assemblages were not affected by physicochemical parameters of water. Important factors shaping the taxonomic composition of leech assemblages were the type of a waterbody (lakes, rivers, ponds) and human impact (including the land use, substratum quality and shoreline type). Waterbody type and human impact affected species richness, with lower values observed in small ponds and under stronger anthropogenic impact. The comparison of assemblages sampled manually from the bottom and from artificial plates deployed in the environment revealed that the artificial plates were a good alternative for traditional methods of leech sampling, particularly at strongly anthropogenically modified locations. Furthermore, several rare species: Alboglossiphonia striata , A. hyalina , Glossiphonia nebulosa , Hirudo medicinalis and Dina sp. were found in the urbanised areas under study, pointing out to their potential role in the conservation of these taxa. [ABSTRACT FROM AUTHOR]

We report the selection in a 15-year-old boy of a multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Aeromonas salmonicida after medicinal leech therapy that required an antibiotic prophylaxis based on piperacillin/tazobactam and cotrimoxazole. Whole genome sequencing of the strain indeed revealed 13 antibiotic resistance genes, including the ESBL CTX-M-3 and the unusual β-lactamase SCO-1. [ABSTRACT FROM AUTHOR]

Diabetic foot ulcer (DFU) with osteomyelitis is the devastating condition, which is a challenge to surgeons in saving the limb of the patient and in many circumstances ends up with amputation, which leaves physical and psychosocial trauma for both the patient and patient's family. A 48-year-old female patient with uncontrolled type 2 diabetes presented with swelling and gangrenous deep circular ulcer of size approx. 3 × 4 cm on plantar aspect of great toe of her left foot with involvement of first webspace from last three months. Plain X ray showed disrupted and necrotic proximal phalanx suggestive of diabetic foot ulcer with osteomyelitis. Despite using antibiotics and antidiabetic drugs for past three months she didn't get significant response and was suggested for toe amputaion. Hence, she approached our hospital for further treatment. We successfully treated the patient with the holistic approach of surgical debridement, medicinal leech therapy (MLT), irrigation of the wound with triphala decoction, jatyadi tail dressings, oral ayurvedic antidiabetic drugs to control blood sugar levels and a mixture of herbo mineral drug which is having antimicrobial property. DFU may lead to infection, gangrene, amputation, death of the patient. Hence it is the need of the hour to look for limb salvage treatment modalities. The holistic approach of these ayurvedic treatment modalities are effective and safe in treating DFUs with osteomyelitis and in preventing amputation. • Osteomyelitis complicated DFU frequently ends up with amputation. • Amputations cause impaired physical function and psychosocial trauma. • Majority of DFUs don't respond to standard treatment. • Interdisciplinary care and multimodal treatments are needed. • Multiple ayurvedic treatments such as surgical debridement, MLT etc. can treat DFU. [ABSTRACT FROM AUTHOR]

Medicinal leeches ( Hirudo verbana ) thermoregulate with respect to their sanguivorous feeding behavior. Immediate postprandial preferences are for warmer than their initial acclimation temperature ( T a , 21 °C, Petersen et al. 2011), while unfed leeches have a lower preferred temperature ( T pref , 12.5 °C). This may reduce energy expenditure and defer starvation if feeding opportunities are limited. Energetic benefits may have an associated cost if low temperatures reduce mobility and the ability to locate further hosts. These costs could be limited if mobility is unimpaired at low temperatures, or if acclimation can restore locomotor performance to the levels at T a . The transition from T a to the unfed T pref significantly reduced speed and propulsive cycle frequency during swimming, and extension and retraction rates during crawling. Aerobic metabolic rate was also reduced from 0.20±0.03 W kg −1 at T a to 0.10±0.03 W kg −1 at T pref . The Q 10 values of 1.7–2.9 for energetic and swimming parameters indicate a substantial temperature effect, although part of the decline in swimming performance can be attributed to temperature-related changes in water viscosity. 6 weeks at T a resulted in no detectable acclimation in locomotor performance or aerobic metabolism. The energetic savings associated with a lower T pref in unfed leeches effectively doubled the estimated time until depletion of energy reserves. Given that some mobility is still retained at T pref , and that acclimation is in itself costly, the energetic benefits of selecting cooler temperatures between feedings may outweigh the costs associated with reduced locomotor performance. [ABSTRACT FROM AUTHOR]

An important question that remains unanswered is how the vertebrate neuroimmune system can be both friend and foe to the damaged nervous tissue. Some of the difficulty in obtaining responses in mammals probably lies in the conflation in the central nervous system (CNS), of the innate and adaptive immune responses, which makes the vertebrate neuroimmune response quite complex and difficult to dissect. An alternative strategy for understanding the relation between neural immunity and neural repair is to study an animal devoid of adaptive immunity and whose CNS is well described and regeneration competent. The medicinal leech offers such opportunity. If the nerve cord of this annelid is crushed or partially cut, axons grow across the lesion and conduction of signals through the damaged region is restored within a few days, even when the nerve cord is removed from the animal and maintained in culture. When the mammalian spinal cord is injured, regeneration of normal connections is more or less successful and implies multiple events that still remain difficult to resolve. Interestingly, the regenerative process of the leech lesioned nerve cord is even more successful under septic than under sterile conditions suggesting that a controlled initiation of an infectious response may be a critical event for the regeneration of normal CNS functions in the leech. Here are reviewed and discussed data explaining how the leech nerve cord sensu stricto ( i.e. excluding microglia and infiltrated blood cells) recognizes and responds to microbes and mechanical damages. [ABSTRACT FROM AUTHOR]

We aimed to assess the effect of Radix Isatidis dietary supplementation on resistance to Proteus mirabilis infection in Poecilobdella manillensis . Six groups of leeches housed in tanks were randomly divided into five treatment groups (fed a basal diet supplemented with 2.5, 5.0, 7.5, 10.0, or 12.5 g/kg of Radix Isatidis ) and one control group (fed a basal diet). Compared with the control group, the specific growth rate (SGR) and weight gain (WG) were significantly decreased in the treatment groups supplemented with Radix Isatidis . However, supplementation with 2.5 to 10.0 g/kg of Radix Isatidis did not produce an effect on mortality. Mortality was recorded after the leeches were challenged with P. mirabilis , and changes were observed in the serum cortisol, glucose, alanine aminotransferase (ALT), 3,5,30-triiodothyronine (T3), thyroxin (T4), alkaline phosphatase (ALP), total protein (TP), globin, and lysozyme contents. In the ingluvies, changes were observed in the total anti-oxidative capacity, superoxide dismutase (SOD) and catalase (CAT) activity, malondialdehyde (MDA) content and heat shock protein 70 (HSP70) gene expression over a period of 7 d. Treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: total anti-oxidative capacity of the ingluvies prior to infection and at 1, 2 and 7 d after infection; serum ALP activity and serum globin concentrations prior to infection and at 1 and 2 d after infection; T4 concentrations prior to infection; serum lysozyme activity at 1 d after infection; SOD and CAT activity in the ingluvies prior to infection and 7 d after infection; T3 concentrations at 1 and 2 d after infection; and the relative level of HSP70 mRNA expression in the ingluvies at 2 d after infection. However, treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: ALT contents prior to infection and 1, 2 and 7 d after infection; serum cortisol concentrations and serum glucose at 1, 2 and 7 d after infection; MDA contents of the ingluvies at 1 and 2 d after infection; and T4 concentrations at 2 and 7 d after infection. Compared with the control group, significantly lower mortality was observed in the groups supplemented with 5.0 to 10.0 g/kg of Radix Isatidis . These results suggest that supplementation with 5.0 to 10.0 g/kg of Radix Isatidis in the basal diet could enhance resistance to P. mirabilis infection in P. manillensis. Statement of relevance We believe that this manuscript is appropriate for publication in Aquaculture because it provides a natural method of controlling disease in P. manillensis , thereby avoiding the use of antibiotics and the development of antibiotic resistance. Furthermore, the results of our study provide new information on the physiological and molecular mechanisms that underlie the protective effect of Radix Isatidis . [ABSTRACT FROM AUTHOR]

Leeches are fascinating creatures: they have simple modular nervous circuitry yet exhibit a rich spectrum of behavioural modes. Leeches could be ideal blue-prints for designing flexible soft robots which are modular, multi-functional, fault-tolerant, easy to control, capable for navigating using optical, mechanical and chemical sensorial inputs, have autonomous inter-segmental coordination and adaptive decision-making. With future designs of leech-robots in mind we study how leeches behave in geometrically constrained spaces. Core results of the paper deal with leeches exploring a row of rooms arranged along a narrow corridor. In laboratory experiments we find that rooms closer to ends of the corridor are explored by leeches more often than rooms in the middle of the corridor. Also, in series of scoping experiments, we evaluate leeches capabilities to navigating in mazes towards sources of vibration and chemo-attraction. We believe our results lay foundation for future developments of robots mimicking behaviour of leeches. [ABSTRACT FROM AUTHOR]

Highlights: [•] ALC was able to block sensitization induced by the administration of serotonin in the leech H. medicinalis. [•] ALC was able to impair behavioural dishabituation induced by the administration of serotonin in H. medicinalis. [•] ALC effect is long lasting being observed up to 11 days after the treatment. [Copyright &y& Elsevier]

Abstract: Although habituation is ubiquitous in the animal kingdom, its underlying mechanisms remain poorly understood. In this study, we began to explore the molecular cascades underlying short-term habituation in the leech Hirudo medicinalis. In H. medicinalis, a training paradigm, consisting of low-frequency repetitive electrical stimulation of the skin, produces a gradual increase in the latency to swim that spontaneously recovers within 20–30min. As first step in determining the molecular pathways in short-term habituation, we examined the role of Ca2+. Both Ca2+ influx through voltage-gated channels and Ca2+ release from intracellular stores were found to contribute to short-term habituation. The analysis of the downstream targets of elevated cytosolic Ca2+ revealed that the activation of the phosholipase A2 was required for the induction of short-term habituation. Finally, we reported that the recruitment of arachidonic acid metabolites, generated by the 5-lipoxygenase pathway, was also necessary for the induction of swim induction habituation. These results provide the framework for a comprehensive characterization of the molecular underpinnings of habituation. This outcome will allow us to compare the mechanisms of habituation with those underlying other forms of nonassociative learning in the leech, such as sensitization and dishabituation, and, more in general, with those governing habituation in different vertebrate and invertebrate model systems. [Copyright &y& Elsevier]

Summary: The nose is a central, prominent unit of the face. The surgical approach to complete nasal amputations is challenging and an experienced microsurgical team should always attempt microvascular replantation. This one-stage procedure could result in a superior clinical outcome compared to delayed reconstructive procedures. Two successful microvascular replantations of the distal half of the nose were performed. Both amputations were the result of two completely different aetiologies (sharp guillotine-like vs. avulsion trauma). However, we were not able to re-establish the venous drainage, which was managed through medicinal leeching. Both replantations resulted in an aesthetically pleasing result and no additional surgery was needed. A microsurgical replantation attempt should always be attempted in nasal amputations and the inability to perform venous anastomosis should not preclude the replant attempt. [Copyright &y& Elsevier]

Summary: Objectives: To evaluate the possible efficacy of medical leeches (Hirudo medicinalis) in the treatment of patients with active osteoarthritis of the knee. Design: Unblinded, randomised controlled trial with outpatients in a crossover design with single interventions of either leeches or transcutaneous electrical nerve stimulation (TENS) as comparator. Main outcome measures: Change in Lequesne''s combined index for pain and function and change (L.I.) and overall assessment of complaints by visual analog scale (VAS). Cross-over at day 42, with further observation period of 21 days. Results: 52 out of 72 screened patients were randomised (intent to treat) to initial treatment with either eight leeches (group 1: 27 patients) or TENS (group 2: 25 patients). Due to phase effects, confirmatory evaluation had to be restricted to the first period. Between days 0 and 21, we observed highly significant (p <0.001) improvements for means of Lequesne''s index from 12.07 to 9.37 and for VAS from 5.89 to 4.16cm for leeches, but no significant differences for TENS. Effect size as group difference was -2.50 for L.I. (95% confidence interval −3.88 to −1.11), resp. −1.86cm for VAS (95% confidence interval −2.85 to −0.87cm). 12 patients (5 group1, 7 group 2) did not finish the trial, mostly due to non-compliance (6). No serious adverse effects were observed. Conclusions: Single leech therapy showed significant, relevant and sustaining effects, comparable to other trials with leeches. The method deserves further research, esp. into mechanisms of possible specific effects and optimization of dosing by number of leeches and possible repeats. [ABSTRACT FROM AUTHOR]

Abstract: Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions. Our group recently demonstrated that the injured leech nerve cord is able to mount an immune response, which promotes the regenerative processes. This defense mechanism is microorganism-specific, suggesting that the leech CNS is able to discriminate among microbial components. We report here the characterization of two receptors potentially implicated in this detection: HmTLR1 and HmNLR. Interestingly, HmTLR1 presents an endosomal distribution in neurons and appears as a chimera combining the mammalian intraendosomal domain of TLR3 and the cytoplasmic section of TLR13, while HmNLR is cytosolic and has the highest homology to NLRC3 receptors. Both receptors show patterns of induction upon stimulation that suggest their involvement in the leech neuroimmune response. This work constitutes the first demonstration in an invertebrate of (i) an intracellular TLR and (ii) a cytosolic PRR related to the NLR family. [Copyright &y& Elsevier]

Abstract: Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal''s midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, α-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals. [Copyright &y& Elsevier]

Purpose: Artery-only fingertip replantation can be reliable if low-resistance flow through the replant is maintained until venous outflow is restored naturally. Injuring the tip of the replant to promote ongoing bleeding augmented with anticoagulation usually accomplishes this; however, such management results in prolonged hospitalization. In this study, we analyzed the outcomes of artery-only fingertip replantation using a standardized postoperative protocol consisting of dextran-40, heparin, and leech therapy. Methods: Between 2001 and 2008, we performed 19 artery-only fingertip replants for 17 patients. All patients had the replanted nail plate removed and received intravenous dextran-40, heparin, and aspirin to promote fingertip bleeding and vascular outflow. Anticoagulation was titrated to promote a controlled bleed until physiologic venous outflow was restored by neovascularization. We used medicinal leeches and mechanical heparin scrubbing for acute decongestion. By postoperative day 6, bleeding was no longer promoted. We initiated fluorescent dye perfusion studies to assess circulatory competence and direct further anticoagulant intervention if necessary. The absence of bleeding associated with an initial rise followed by an appropriate fall in fluorescent dye concentration would trigger a weaning of anticoagulation. Results: All of the 19 replants survived. The average length of hospital stay was 9 days (range, 7–17 d). Eleven patients received blood transfusions. The average transfusion was 1.8 units (range, 0–9 units). All patients were happy with the decision to replant, and the cosmetic result. Conclusions: A protocol that promotes temporary, controlled bleeding from the fingertip is protective of artery-only replants distal to the distal interphalangeal joint until physiologic venous outflow is restored. The protocol described is both safe and reliable. The patient should be informed that such replant attempts may result in the need for transfusions and extended hospital stays, factors that can help the physician and patient decide whether to proceed with repantation. Type of study/level of evidence: Therapeutic IV. [Copyright &y& Elsevier]

Clin Microbiol Infect 2010; 16: 563–567 The use of the medicinal leech ( Hirudo medicinalis) in promoting venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially in plastic and reconstructive surgery, has been complicated by infections caused by Aeromonas spp. These are leech endosymbionts for which patients undergoing hirudotherapy frequently receive systemic chemoprophylaxis. In order to evaluate the possibility of rendering leeches safe for use on patients, H. medicinalis were fed artificially with a 2 g/L arginine solution (used as a phagostimulant) supplemented with ciprofloxacin (100 mg/L). Aeromonads were detected in 57 out of 80 control leeches (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001). Treated leeches survived for up to 4 months. Tested weekly, 61% of these leeches took human blood for at least 4 weeks after treatment and all remained negative for aeromonads. All water samples in which leeches were kept before treatment were contaminated with Aeromonas spp.; none were detected in any of the NaCl/arginine solutions with which treated animals were fed. Molecular characterization of two phenotypically distinct isolates using gyrB sequencing showed that one clustered tightly with A. veronii and the other was closely related to A. media. Other environmental bacteria and fungi were isolated from 26.5% of treated leeches that had taken a blood meal 1–4 weeks after treatment. Ciprofloxacin reduced the number of leech-associated aeromonads to undetectable levels for extended periods. Most treated leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy. [ABSTRACT FROM AUTHOR]

Many postoperative complications have been reported after repair of classic bladder exstrophy. We present a case of medicinal leech therapy for glans penis congestion following exstrophy repair in an infant. A 2-week-old male with classic bladder exstrophy underwent complete primary repair. On postoperative day 1, he developed rapidly worsening glans penis venous congestion. Medicinal leech therapy was instituted with antibiotics and blood transfusions to maintain a hematocrit >30%. After 24 hours, venous congestion improved and therapy was discontinued. The patient's remaining hospital course was uncomplicated. Medicinal leeches are an effective therapy to relieve glans penis venous congestion. [ABSTRACT FROM AUTHOR]

Abstract: Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. Opioids have been postulated to modulate various parameters of the immune response. Morphine and leech morphine-like substance are shown to release NO and suppress microglial activation. Regarding the known immuno-modulatory effects of selective μ and κ ligands, we have assessed the effect of these agents on accumulation of microglia at the site of injury in leech CNS. Leech nerve cords were dissected, crushed with fine forceps and maintained in different concentrations of opiates in culture medium for 3 h and then fixed and double stained with Hoechst 33258 and monoclonal antibody to endothelial nitric oxide synthase (NOS). Morphine and naloxone (≥10−3 M) but not selective μ agonist, DAMGO [d-Ala2, N-Me-Phe-Gly5(ol)-enkephalin] and antagonist, CTAP [D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] inhibited the microglial accumulation. The effect of morphine was abrogated by pre-treatment with naloxone and also non-selective NOS inhibitor, l-NAME [Nω-nitro-l-arginine-methyl-ester; 10−3 M] implying an NO-dependent and μ-mediated mechanism. These results are similar to properties of recently found μ-3 receptor in leech, which is sensitive to alkaloids but not peptides. Both selective κ agonist, U50,488 [3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)-benzeneacetamide; ≥10−3 M], and antagonist, nor-binaltorphimine (nor-BNI; ≥10−3 M), inhibited the accumulation. The effect of nor-BNI was reversed by l-NAME. Immunohistochemistry showed decreased endothelial NOS expression in naloxone and U50,488-treated cords. Since, NO production at the injury site is hypothesized to act as a stop signal for microglias, opioid agents may exert their effect via changing of NO gradient along the cord resulting in disruption of accumulation. These results suggest an immuno-modulatory role for μ and κ opioid receptors on injury-induced microglial accumulation which may be mediated via NO. [Copyright &y& Elsevier]

Abstract: The medicinal leech has served as an important experimental preparation for neuroscience research since the late 19th century. Initial anatomical and developmental studies dating back more than 100 years ago were followed by behavioral and electrophysiological investigations in the first half of the 20th century. More recently, intense studies of the neuronal mechanisms underlying leech movements have resulted in detailed descriptions of six behaviors described in this review; namely, heartbeat, local bending, shortening, swimming, crawling, and feeding. Neuroethological studies in leeches are particularly tractable because the CNS is distributed and metameric, with only 400 identifiable, mostly paired neurons in segmental ganglia. An interesting, yet limited, set of discrete movements allows students of leech behavior not only to describe the underlying neuronal circuits, but also interactions among circuits and behaviors. This review provides descriptions of six behaviors including their origins within neuronal circuits, their modification by feedback loops and neuromodulators, and interactions between circuits underlying with these behaviors. [Copyright &y& Elsevier]

Acetyl-l-carnitine is known to improve many aspects of the neural activity even if its exact role in neurotransmission is still unknown. This study investigates the effects of acetyl-l-carnitine in T segmental sensory neurons of the leech Hirudo medicinalis. These neurons are involved in some forms of neural plasticity associated with learning processes. Their physiological firing is accompanied by a large afterhyperpolarization that is mainly due to the Na+/K+ ATPase activity and partially to a Ca2+-dependent K+ current. A clear-cut hyperpolarization and a significant increase of the afterhyperpolarization have been recorded in T neurons of leeches injected with 2 mM acetyl-l-carnitine some days before. Acute treatments of 50 μM acetyl-l-carnitine induced similar effects in T cells of naive animals. In the presence of apamin, a pharmacological blocker of Ca2+-dependent K+ channel, acetyl-l-carnitine still enhanced the residual afterhyperpolarization, suggesting an effect of the drug on the Na+/K+ATPase. Acetyl-l-carnitine also increased the hyperpolarization induced by intracellular injection of Na+ ions. Therefore, acetyl-l-carnitine seems to be able to exert a positive sustained effect on the Na+/K+ ATPase activity in leech T sensory neurons. Moreover, in these cells, widely arborized, the afterhyperpolarization seems to play an important role in determining the action potential transmission at neuritic bifurcations. A computational model of a T cell has been previously developed considering detailed data for geometry and the modulation of the pump current. Herein, we showed that to a larger afterhyperpolarization, due to the acetyl-l-carnitine-induced effects, corresponds a decrement in the number of action potentials reaching synaptic terminals. [Copyright &y& Elsevier]

We report here some results of a proteomic analysis of changes in protein expression in the leech Hirudo medicinalis in response to septic injury. Comparison of two-dimensional protein gels revealed several significant differences between normal and experimental tissues. One protein found to be up-regulated after septic shock was identified, through a combination of Edman degradation, mass spectrometry, and molecular cloning, as a novel member of the hemerythrin family, a group of non-heme-iron oxygen transport proteins found in four invertebrate phyla: sipunculids, priapulids, brachiopods, and annelids. We found by in situ hybridization and immunocytochemistry that the new leech protein, which we have called neurohemerythrin, is indeed expressed in the leech central nervous system. Both message and protein were detected in the pair of large glia within the ganglionic neuropile, in the six packet glia that surround neuronal somata in each central ganglion, and in the bilateral pair of glia that separate axonal fascicles in the interganglionic connective nerves. No expression was detected in central neurons or in central nervous system microglia. Expression was also observed in many other, non-neuronal tissues in the body wall. Real-time PCR experiments suggest that neurohemerythrin is up-regulated posttranscriptionaly. We consider potential roles of neurohemerythrin, associated with its ability to bind oxygen and iron, in the innate immune response of the leech nervous system to bacterial invasion. [ABSTRACT FROM AUTHOR]

In this paper the role of serotonin (5HT) and cyclic AMP (cAMP) in sensitization and dishabituation of swim induction (SI) has been investigated in the leech Hirudo medicinalis. Electrical stimulation of the body wall evokes swimming activity with a constant latency. In animals with a disconnection between head ganglion and segmental ganglia, repetitive stimulation induces habituation of swimming whereas brushing on the dorsal skin provokes sensitization of a naïve response or dishabituation of a previously habituated response. Our findings indicate that 5HT is the neurotransmitter underlying both sensitization and dishabituation of SI. Injection of the 5HT receptor blocking agent methysergide impaires the onset of sensitization and dishabituation induced by brushing. Moreover, injection of 5HT mimics these forms of nonassociative learning, whereas injection of dopamine does not. Finally, the effects of 5HT are mediated by cAMP: (1) after injections of specific adenylate cyclase inhibitors such as MDL 12.330A or SQ22536, brushing becomes ineffective in facilitating the SI in either non-habituated or habituated animals. (2) 8Br-cAMP application mimics both sensitization and dishabituation of SI. [Copyright &y& Elsevier]

Release of neurotransmitter from presynaptic nerve terminals is mediated by SNARE proteins, which are located on the vesicle and plasma membranes. These proteins form a SNARE complex thought to mediate membrane fusion. Complexin is a soluble protein essential for transmitter release, which has been postulated to bind to and stabilise the SNARE complex. We have cloned a complexin homologue, Hm-cpx1, from the leech, Hirudo medicinalis. This protein is expressed in only a subset of neurons in the leech CNS, including the Retzius and P neurons. It is 33% identical to rat complexin I, and 44% identical to squid complexin. Sequence conservation is particularly high in the predicted SNARE binding domain. [Copyright &y& Elsevier]

Over the past decade, several putative homologs of cannabinoid receptors (CBRs) have been identified by homology screening. Homology screening utilizes sequence alignment search engines to recognize homologs. We investigated these putative CBR homologs further by ‘functional mapping’ of their deduced amino acid sequences. The entire pharmacophore of a CBR has not yet been elucidated, but point-mutation studies have identified over 20 amino acid residues that impart CBR specificity for ligand recognition and/or signal transduction. Twenty point-mutation studies were used to construct a CBR functionality matrix. Sixteen putative CBR homologs were then mapped over the matrix. Several putative homologs did not hold up to this analysis: human GPR3, GPR6, GPR12, and Caenorhabditis elegans C02H7.2 expressed a series of crippling substitutions in the matrix, strongly suggesting they do not encode functional CBRs. Mapping the contested leech (Hirudo medicinalis) CBR sequence suggests that it encodes a functional CB1; it expresses fewer substitutions than the sea squirt (Ciona intestinalis) CB1 sequence. Mapping a putative CB2 ortholog in the puffer fish (Fugu rubripes T012234) suggests it may encode a CBR other than CB2. These findings are consistent with the lack of experimental data proving these putative CBRs have affinity for cannabinoid ligands. Matrix analysis also reveals that SR144528, a ‘CB2-specific’ synthetic antagonist, has affinity for non-mammalian CB1 receptors, and that L3.45 appears to be CB2-specific, its cognate in CB1 receptors is F3.45. In conclusion, functional mapping, utilizing point-mutation studies, may improve the specificity of homology screening performed by sequence alignment search engines. [Copyright &y& Elsevier]

Behaviour has been shown to be a sensitive indicator of chemically induced stress and pathology in aquatic organisms. However, ecotoxicological investigations on medicinal leech are restricted because of scarce leech resources in natural waters. We used artificially bred medicinal leech (Hirudo medicinalis L.) of two different ages: young (1–2 weeks old) and adult leeches (1 year old). Animals were exposed to: (1) waters of Lake Drukshiai—the cooler of Ignalina Nuclear Power Plant, (2) sediments of the river Nemunas and (3) solution of heavy metal model mixture (HMMM). The following behavioural responses of leech were investigated: mobility (number of moving individuals within certain periods of time), avoidance response (number of individuals escaping the tested water or sediments) changes in body shape (contractions of some muscles, abnormal position of suckers) and feeding activity (longevity of attachment process, interruptions of feeding bouts, size of blood meal). Mobility of young leeches was increased in the tested waters of Lake Drukshiai, whereas adult leeches showed no changes of this index. Avoidance response as well as impaired feeding activity (prolonged attachment process or completely suppressed attachment reflex, decreased size of blood meal) was observed in young leeches exposed to the waters from Lake Drukshiai and in adult animals exposed to all three kind of trials mentioned above. Avoidance response and changes in mobility were recorded during the first hour of exposure to tested samples; therefore, these responses can be used as an express method for water and sediment pollution assessment. Impaired feeding activity was recorded after 1–3-week exposure: this response may be used in assessing a chronic toxicity of pollution. Medicinal leech, due to its sensitivity, simplicity of measured indices and ease of laboratory maintenance, can be used as a test organism in ecotoxicity studies. [Copyright &y& Elsevier]

A myomodulin peptide has been suggested to mediate the response of the giant glial cells to stimulation of the Leydig interneuron in the central nervous system of the leech Hirudo medicinalis [Eur. J. Neurosci. 11 (1999) 3125]. We have now studied the glial response to the endogenous leech MM peptide (GMGALRL-NH2, MMHir). The peptide evokes a membrane outward current (EC50 ∼2 μM), which neither desensitizes nor shows any sign of run-down, and elicits a K+ conductance increase of the glial cell membrane. The peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) enhances the glial current response, suggesting the presence of endogenous extracellular peptidases. [Copyright &y& Elsevier]

The electrogenic Na+ absorption across tight epithelia from invertebrates follows the principles analog to the mechanisms found in vertebrates. Extracellular Na+-ions pass the apical cell membranes through highly selective Na+ channels and follow an electrochemical gradient which is sustained by the basolateral Na+/K+-ATPases. These apical Na+ channels are selectively blocked by amiloride and represent the rate-limiting target for the control of transcellular Na+ uptake. Although annelids express ADH-like peptide hormones, they lack the osmoregulatory mineralocorticoid system with the vertebrate-specific key hormone aldosterone. Thus, their epithelia may represent interesting models for investigation of ion transport regulation. While the formation of urine in the nephridia of, for example, leeches had been subject to intensive studies, the investigation of ion transport across their body wall was largely neglected. We use dissected segments of integuments from the limnic leech Hirudo medicinalis and, recently, from the earthworm Lumbricus terrestris for Ussing chamber experiments. We investigate transintegumental ion transport with focus on control of electrogenic Na+ uptake and the amiloride-sensitive part of it and identified several extracellular factors as peptide hormones, tri- and divalent cations or purinergic molecules with regulatory effects on it. Meanwhile, there exists a macroscopic view on Na+ absorption; however, other ion transport mechanisms across annelid integuments still await scientific effort. Here we present a concise synopsis about the electrophysiology of annelid integuments to illustrate the state of science and to evaluate whether further studies in this particular field may be of interest. [Copyright &y& Elsevier]

• ASFV can spread in the environment and infect pigs via water and food, mostly through ingestion and occasionally through skin contact. • ASFV is able to survive in several environments, notably in water, food and soil. • The aim was to study the ability of leeches to persistently harbor ASFV. • Also leeches excrete the virus, we observed the features of this excretion, as well as the persistence of indefectibility of the excreted viruses. This research is focused on the study of African swine fever virus (ASFV) survival in the leech Hirudo medicinalis. To detect the infection route of the virus in leeches, two introduction methods were used: blood-feeding from infected swine hosts and exposure to ASFV-contaminated water (leeches cultivated with ASFV). This study found that the survival of ASFV in leeches was much longer than that in leech-free medium. The persistent presence of the virus in leeches and leech excretion was detected in both groups. The virus excreted from both groups of leeches in the terminal stages of the experiment was able to induce productive infection in porcine alveolar macrophages. In an attempt to understand ASFV, transmission to pigs was conducted through the ingestion of leech-cultivated water and leeches that had fed on ASFV-infected animals or that were cultivated environmentally with the virus. The ingestion of some samples at 60–80 days after cultivation demonstrated successful ASFV transmission via per os infection. In conclusion, leeches can serve as a possible reservoirs for ASFV in the absence of its main hosts – pigs and some ticks of the genus Ornithodoros. [ABSTRACT FROM AUTHOR]

Summary Introduction Medicinal leeches have been part of the therapeutic armamenterium of plastic surgeons for more than 50 years. While their use in hand surgery is a matter of course, their use in salvage of flaps with venous congestion remains facultative depending on teams. Materials and methods We conducted a systematic review of leech therapy for flap salvage between 1960 and 2015, analyzing 121 articles and subsequently taking into consideration 41 studies. In parallel, we collected data from 43 patients for whom leach therapy had recently been applied in treatment of venous insufficiency in pedicled or free flaps after revision surgery had failed to improve flap vascularization, or in cases where flap revision was not appropriate. The data collected pertained to relevant indications, treatment procedure, efficacy, adjuvant therapies, side effects and complications. Results For this indication, the success rate of leech therapy ranged from 65 to 85% (83.7% in our series) according to the situations encountered. Optimal frequency of application ranged from 2 to 8 hours, while average overall duration ranged from 4 to 10 days. The number of leeches to be applied can be determined depending on volume of the flap. In 50% of the cases reported in the literature, the patients required transfusion. Antibiotic prophylaxis against Aeromonas is highly advisable. A ciprofloxacin and trimethoprim-sulfametoxazole combination currently appears as the most relevant prophylactic antibiotherapy. Conclusion Hirudotherapy is a reliable treatment in cases of patent venous insufficiency of pedicled or free flaps (or when revision surgery is not recommended). Even though the relevant literature is highly heterogeneous, we have attempted to put forward a specific protocol bringing together dosage, delivery route, frequency of administration and appropriate prophylactic antibiotherapy. An algorithm for treatment and management of venous congestion and a practical information sheet have been placed at the disposal of plastic surgery teams. [ABSTRACT FROM AUTHOR]

Summary: Medicinal leeches (Hirudo medicinalis) are commonly used in plastic surgery for the salvage of congested flaps and replanted parts compromised by venous congestion. Infection associated with leech therapy is a documented complication of leech application, with reported incidences ranging from 2.4 to 20% [De Chalain TM. Exploring the use of the medicinal leech: a clinical risk-benefit analysis. J Reconstr Microsurg 1996;12(3):165–72.]. We describe a case of delayed leech-borne infection, from the escharotic portion of a latissimus dorsi flap, which developed several days after stopping leech therapy for venous congestion in a reconstructed breast. [Copyright &y& Elsevier]