Design and Optimization of a Novel-Herbosomal Loaded PEG-Poloxamer Topical Formulation For the Treatment of Cold Injuries: A Quality By Design Approach

Cold injury/injuries can range from minor chilblain to extreme form of frostbite. Cold injuries are pathologically a combination of ice crystal formation in tissue with inflammation, thrombosis and ischemia to extremities, necessitating limb amputation in extreme cases due to tissue necrosis. Less severe forms of cold injuries can be managed by gentle rewarming of limb and avoiding exposure to cold leading to favorable outcomes, however severe forms of frostbite are a cause of major concern to patients as well as the treating physician. Due to lack of effective pre-treatment modalities and paucity of research in prophylaxis and therapeutics of cold injuries, we have developed a novel-herbosomal loaded PEG-Poloxamer topical formulation (n-HPTF) by Quality by Designed approach, incorporating natural ingredients which are having potential therapeutic effect for the treatment of cold injury in a form of a novel-lipid vesicles (herbosomes) loaded in polymers (PEG-3350 and Poloxamer-188) resulting excellent occlusive barrier and thus promote rapid healing. Optimized novel-herbosomes showed entrapment efficiency > 90% and < 300 nm mean particle size and in-vitro drug permeation of about 2 µg/cm2 followed by Higuchi’s release kinetic. Skin irritancy study on female Sprague Dawley rats showed no edema or erythema. In-vivo bio-efficacy study was performed and found significantly good at p-value < 0.05 when compared to the standard treatment groups.

Nonfreezing Cold Injury (Trench Foot)

Nonfreezing cold injury (NFCI) is a modern term for trench foot or immersion foot. Moisture is required to produce a NFCI. NFCI seldom, if ever, results in loss of tissue unless there is also pressure necrosis or infection. Much of the published material regarding management of NFCIs has been erroneously borrowed from the literature on warm water immersion injuries. NFCI is a clinical diagnosis. Most patients with NFCI have a history of losing feeling for at least 30 min and having pain or abnormal sensation on rewarming. Limbs with NFCI usually pass through four ‘stages.’ cold exposure, post-exposure (prehyperaemic), hyperaemic, and posthyperaemic. Limbs with NFCI should be cooled gradually and kept cool. Amitriptyline is likely the most effective medication for pain relief. If prolonged exposure to wet, cold conditions cannot be avoided, the most effective measures to prevent NFCI are to stay active, wear adequate clothing, stay well-nourished, and change into dry socks at least daily.

Potential Roles for Pattern Molecule of PAMP-triggered Immunity in Improving Crop Cold Tolerance

Pathogen infection cross-activates cold response and increase cold tolerance of host plants. However it is not possible to use the infection to increase cold tolerance of field plants. Here flagellin 22 (flg22), the most widely-studied PAMP, was used to mimic the pathogen infection to cross-activate cold response. Flg22 treatment alleviated the injury caused by freezing in Arabidopsis, oilseed and tobacco. In Arabidopsis, flg22 activated the expression of immunity and cold-related genes. Moreover the flg22 induced alleviation of cold injury was lost in NahG transgenic line (SA-deficient), sid2-2 and npr1-1 mutant plants, and flg22-induced expression of cold tolerance-related genes, which indicating that salicylic acid signaling pathway is required for the alleviation of cold injury by flg22 treatment. In short flg22 application can be used to enhance cold tolerance in field via a salicylic acid-depended pathway.

Complications classification of local cold injury

The OBJECTIVE of this work was to develop a classification of complications of local cold injury.METHODS AND MATERIALS. The study is based on the results and analysis of the treatment of 132 patients with local cold injury of the II–IV degree of the lower extremities. The victims were hospitalized in the regional center for thermal trauma on the basis of the «City Clinical Hospital № 1» in the period from 2018 to 2019. Post-traumatic complications were detected in 54 patients (40.1 %), of which 32 developed early and 22 patients developed late complications. The remaining 78 patients after discharge from the hospital did not seek medical help again and were under the supervision of a polyclinic surgeon.RESULTS. In the course of the study, it was found that in 32 out of 132 patients in the early period of local cold injury of the lower extremities, sensitivity disorders, muscle weakness, convulsions, impaired coordination of movements, graft rejection, wound suppuration and suture failure, stump necrosis were detected in the affected segments of the extremities. 22 victims developed late complications of local cold injury: trophic ulcers of the stumps of the feet, osteomyelitis with the formation of sequesters, gangrene of the stumps of both feet. As a result of the study and pathogenetic interpretation of the consequences of cryoinjury, a new classification of complications of local cold injury was formed and the criteria for the forms of «cold extremity» were identified.CONCLUSION. Post-traumatic complications were detected in almost half of the victims with local cold injury (54 patients, 40.1 %). Thus, in the early stages of cryoinjury, 32 patients were found to have impaired sensitivity of the affected limb, muscle weakness, convulsions, and impaired coordination of movements; 4 (12.5 %) had treatment complicated by graft rejection; 5 (15.5 %) had wound suppuration and suture failure; 2 (6.25 %) had stump necrosis. In the late period, complications of local cold injury were registered in 22 patients. Of these, 12 (55 %) patients were found to have trophic disorders; osteomyelitis was detected in 8 (36 %) patients; in 2 (9 %) — gangrene of the distal segment of the affected limb.

Capsaicin 8% Patch Treatment in Non-Freezing Cold Injury: Evidence for Pain Relief and Nerve Regeneration

Introduction: Neuropathic pain associated with Non-freezing Cold Injury (NFCI) is a major burden to military service personnel. A key feature of NFCI is reduction of the intra-epidermal nerve fibre density in skin biopsies, in keeping with painful neuropathy. Current oral treatments are generally ineffective and have undesirable side effects. Capsaicin 8% patch (Qutenza) has been shown to be well-tolerated and effective for reducing neuropathic pain, for up to 3 months after a single 30-minute application.Methods: In this single-centre open label study, 16 military participants with NFCI (mean duration 49 months) received 30-minute Capsaicin 8% patch treatment to the feet and distal calf. Pain symptoms were assessed using a pain diary (with the 11-point Numerical Pain Rating Scale, NPRS) and questionnaires, the investigations included skin biopsies, performed before and three months after treatment.Results: Participants showed significant decrease in spontaneous pain (mean NPRS: −1.1, 95% CI: 0.37 to 1.90; p = 0.006), and cold-evoked pain (−1.2, 95% CI: 0.40 to 2.04; p = 0.006). The time-course of pain relief over 3 months was similar to other painful neuropathies. Patient Global Impression of Change showed improvement (p = 0.0001).Skin punch biopsies performed 3 months after the patch application showed significant increase of nerve fibres with structural marker PGP9.5 (intra-epidermal nerve fibres [IENFs], p &lt; 0.0001; sub-epidermal nerve fibres [SENFs]; p =&lt; 0.0001), and of regenerating nerve fibres with their selective marker GAP43 (p = 0.0001). The increase of IENFs correlated with reduction of spontaneous (p = 0.027) and cold-evoked pain (p = 0.019).Conclusions: Capsaicin 8% patch provides an exciting new prospect for treatment of NFCI, with regeneration and restoration of nerve fibres, for the first time, in addition to pain relief.

Comprehensive assessment of peripheral innervation and microcirculatory function in local cold trauma

Актуальность криопоражений обусловлена большим числом пострадавших, сложностью лечения, длительной утратой трудоспособности, тяжелыми последствиями и, как следствие, высоким уровнем инвалидизации. Цель исследования -- оценка изменений микрокровотока и периферической иннервации при местной холодовой травме и формирование прогностической модели уровня повреждения конечности. Методика. Под наблюдением было 35 больных с местной холодовой травмой нижних конечностей III-IV степени. Использован неинвазивный метод лазерной доплеровской флуометрии с помощью аппарата ЛАКК-02, а также метод накожной электронейромиографии с помощью аппарата Нейро-ВМП, компании Нейрософт. Результаты. Несмотря на гипотетически функциональную связь между микрокровотоком кожи и уровнем отморожения пораженной конечности, статистически значимых результатов получено не было (p > 0,05), что свидетельствует о более сложном генезе патологических изменений в пораженной конечности. Между тем, у пострадавших с местной криотравмой отмечены выраженные изменения периферической иннервации. Нейропатия прогрессирует с увеличением уровня повреждения. Заключение. У пациентов с местной холодовой травмой отмечается снижение биоэлектрической активности мышц в области пораженной конечности. Cryotrauma is relevant due to a large number of patients, the complexity of treatment, the likelihood of severe complications, and long-term disability. The aim was to assess oscillations in microcirculation and peripheral innervation during regional cold injury, and to form a prognostic model according to the severity of limb injury. Methods. Studies were carried out in 35 patients with III-IV degree local cold injury of a leg. Non-invasive laser Doppler flowmetry was performed with a LAKK-02 device, and peripheral innervation was evaluated by cutaneous electroneuromyography with a Neurosoft Neuro-VMP device. The patients were divided into 3 groups depending on the injury. Results. Despite the hypothetical, functional relationship between skin microcirculatory blood flow and the degree of frostbite of the affected tissue, no statistically significant association was detected (p > 0.05), which indicates a more complex genesis of the cold-induced pathology. However, there were pronounced changes in neural activity in this tissue. Neuropathy increased with increasing injury. Conclusions. In patients with local cold trauma there is a decrease in the bioelectric activity of the muscles in the damaged tissue.

Non-obvious effects of montelukast – leukotriene receptor blocker: frigoprotective and anticonvulsant properties

The participation of arachidonic acid metabolism products – prostaglandins and leukotrienes – in the process of inflammation is a common pathogenetic link of cold injury and epilepsy. Montelukast is widely used for the treatment of bronchial asthma and allergic rhinitis as a leukotriene receptor blocker. However, the mechanism of action of the drug suggests a wider range of its pharmacological properties and the corresponding scope of application. This study is aimed to determine the effectiveness of montelukast as a potential frigoprotective and anticonvulsant drug. Experiments were performed on 73 white mice weighing 20-22 g on models of acute general cooling and pentylenetetrazol convulsions. Frigoprotective properties were studied at a temperature of –18°C, recording the lifetime. Montelukast ("Singular", 2 mg/kg), acetylsalicylic acid ("Aspirin", 50 mg/kg), celecoxib ("Celebrex", 74 mg/kg), diclofenac sodium ("Voltaren", 14 mg/kg) were administered intragastrically as a suspension in a prophylactic mode, 30 minutes before the cold injury. In the study of anti­convulsant activity, montelukast ("Singular", 4 mg/kg) and sodium valproate ("Depakin", 300 mg/kg) were admi­nistered intragastrically 30 minutes before stimulating convulsions by subcutaneous administration of pentylene­tetrazole (90 mg/kg). The latent period of convulsions, the number of convulsions per 1 animal, % of mice with clonic and tonic paroxysms, the severity of convulsions in points, the duration of the convulsive period, the lifetime of animals and lethality were recorded for an hour. On the model of acute general cooling, montelukast showed a dose-dependent frigoprotective effect at a dose of 2 mg/kg surpassing drugs with proven frigoprotective properties – acetylsalicylic acid and celecoxib. On the model of pentylenetetrazole-induced convulsions, montelukast statistically significantly reduced the integral indicator of anticonvulsant activity – lethality – by 2.57 times. Thus, the experiment proved the significant role of leukotrienes in the pathogenesis of cold injury and epilepsy and justified the feasibility of further study of the frigoprotective and anticonvulsant properties of montelukast – leukotriene receptor blocker a drug as for adjuvant therapy, especially when these pathologies are combined with bronchial asthma and allergic rhinitis.

Improved Cold Tolerance is Associated with Altered Cell Wall Composition in Pre-Planted Tobacco

Tobacco seedlings produced by floating system are susceptible to cold injury. This problem can be solved by pre-planting technique. However, little is known about the mechanism underlying the improved cold tolerance in pre-planting tobacco. To investigate it, the cytological features of the leaf and root cells from floating and pre-planting tobacco were studied. Obvious cold injury and rupture of cell membrane system were observed in the cells from floating tobacco under cold stress conditions but very little in the cells from pre-planting tobacco. The cell wall in cells from pre-planting tobacco was thicker than in floating tobacco before cold stress. The cell wall was higher in cellulose and pectin contents in pre-planting tobacco than in floating tobacco before and after cold stress. These results suggest that pre-planting technique facilitates the accumulation of cell wall compositions-cellulose and pectin in pre-planting plants to prevent the cold injury, and possibly attenuates the negative effect of flooding stress on cell wall composition. © 2021 Friends Science Publishers