Development of high-precision automated dynamic plantar aesthesiometer (ADPA): a promising tool in pain research

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To develop novel analgesics, in vivo preclinical assessment of the pain threshold is inevitable. Investigation of the nociception in rodents is still challenging, since most of the currently available methods are manually operated. So, the results highly depend on the experience of the examiner and can be significantly biased by subjective human factors. To improve this translational research paradigm, advanced tools are needed in this field. Therefore, the aim of the present study was to develop a new generation automated pain assessment device. In collaboration with Z-Elektronika Ltd., Pécs, Hungary we have designed and validated high-precision automated dynamic plantar aesthesiometer (ADPA) that is suitable for the assessment of mechanonociceptive threshold in rats and mice. It utilizes artificial intelligence (AI) to automatically recognize the animals investigated. The system's software controls the mechanical stimulation of the hindpaws with simultaneous video recording of the nocifensive reaction and analysis of the pain thresholds. The main advantage of ADPA is the automated, computer-controlled induction and evaluation of the pain threshold, increasing the quality, comparability, reproducibility, and objectivity of the results. This device may significantly enhance the accuracy of pain assessment in animal models and contribute to improved preclinical pain research. Biological sciences/Biological techniques Biological sciences/Drug discovery Physical sciences/Engineering Health sciences/Medical research Biological sciences/Neuroscience pain measurement mechanonociceptive threshold novel automated device Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction As the International Association for the Study of Pain (IASP) defined, pain is an unpleasant sensory and emotional experience linked to actual or potential tissue damage that affects individuals all over the world [ 1 ]. Acute pain is adaptive and serves a protective function by signalling potential or avoiding actual tissue damage. If pain persists for weeks to months, the initial acute pain becomes chronic, loses its protective function and becomes maladaptive [ 2 ]. Approximately 20% of the population suffer from chronic pain, with 8% reporting a negative impact on daily life [ 3 ]. Chronic pain associated with neuroplastic changes in the nervous system, induces significant impairment in quality of life and increased healthcare cost as well as huge socio-economic burden for the government of the countries [ 4 , 5 ]. Pain and its impact vary among individuals and are influenced by environmental, cultural, and lifestyle factors [ 6 ]. Since animals could not communicate the pain verbally; only the nocifensive reaction (spinal reflexes) evoked by painful stimuli can be measured. Development of a sensitive, objective equipment which can be detect any signs of pain together with the nociceptive responses is challenging. In preclinical animal studies, the most frequently used tools work by measuring the rodents’s nocifensive movements (paw withdrawal; shaking, lifting or licking of the affected paws). The harmful stimulus can be either mechanical (Dynamic Plantar Aestesiometer (DPA), Ugo Basile, Italy) [ 7 – 11 ]; Von Frey filament [ 12 – 15 ], and the Randall-Selitto analgesimeter [ 16 – 18 ], or heat using increasing/decreasing temperature hot/cold plate or water bath [ 19 ], Hargreaves apparatus [ 20 ]. Beside these challenging factors, the currently available methods have several other limitations, e.g. most of them are manually operated, moreover, some of them require that the animals should be held in the examiners hand throughout the whole examination time, which is highly stressful for the animals. Even changing the person of the examiner during the experiment, the unfamiliar smell or different mood status can be also stressful for the animals. This stress alone can modify the measurement outcomes, so the results obtained contain many subjective elements and depend heavily on the experience of the examiner, and even result in low standardization, which can reduce the validity of the data and lead to incorrect conclusions. In addition, this subjectivity can reduce the validity of the measured data and lead to false conclusions. Therefore, the development of pain measurement tools is crucial for collecting objective, reliable data, measuring effectively the pain intensity, detecting the efficacy and dosages of the novel therapeutic agents. Improvement of the pain detecting device helps to minimalize the numbers and the suffering of the animals used in preclinical studies and can elevate the translational value of this preclinical research further increasing the successfulness of analgesic drug research. Based on this, our experiments aimed to develop a new-generation pain threshold assessment device that automates both the delivery of mechanical stimuli and the evaluation of nocifensive behaviour, thereby reducing human bias and improving the reproducibility and reliability of pain assessment. Furthermore, we also aimed to validate our newly developed equipment in preclinical animal models of acute and chronic pain, for which the determination of mechanical pain threshold and mechanical hyperalgesia/allodynia is essential, and to compare these data to those gained with conventional DPA. Methods Development process of the high-precision automated dynamic plantar aesthesiometer (ADPA) In the first part of the project, in collaboration with Z-Elektronika Ltd. we have constructed a new generation mechanical pain threshold measurement apparatus in which the mechanical stimulation of the hindpaw as well as the detection and analysis of the nocifensive behaviour is driven automatically by the computer connected to the equipment. This high-precision automated dynamic plantar aesthesiometer (ADPA) consists of three separated well-ventilated and well-illuminated plastic compartments placed on a raised metal grid. The size of these compartments is 20 cm x 20 cm and allows the parallel measurement of three rats. Moreover, each compartment can be further divided into four parts, and the 10 cm x 10 cm parts allow to measure 12 mice altogether, so the equipment is suitable for the investigation of both rodent strains that are routinely applied in preclinical studies. The equipment consists of 4 cameras, three of which serve the detection of the position and posture of the animal from above, in order to ensure the precise moving of the needle, while the fourth camera is built in the actuator unit and it is responsible for the videorecording of the nocifensive behaviour from below. The linear actuator unit is found below the metal grid, including the blunt-end needle exerting the mechanical stimulation of the hindpaw. The moving of the linear actuator unit and the elevation of the needle is controlled by the system’s software based on the videorecorded alignment of the animal. At the bottom of the equipment, a removable tray is found enabling easy and fast cleaning of the device, moreover, the metal grid is exchangeable allowing fast changeover between the investigation of different strains (Fig. 1 A). The apparatus is connected to a computer, that runs a user-friendly, artificial intelligence (AI)-driven software controlling the movement of the actuator unit and the needle, as well as performing the video analysis of the nocifensive behaviour. Before starting the measurement, the investigator can choose from normal, control and manual modes, as well as perform several settings, including the strain, sex, treatment and individual identifier of the animal, the minimum body length, the target leg (left or right), the maximum target force, the minimum force (that is only applicable for control measurements), the force time, the number of measurements to be taken per hindlimb, the animal’s relax time, the time between measurements, as well as the waiting time after start or the last stop. The device provides the results of the measurements in an individual data table for each animal including the actual maximum measured force, the target maximum force and the measurement number, alongside the video recordings of the measurement from the top and bottom cameras (Fig. 1 /B-C). These settings allow the automated, real-time measurement of mechanonociceptive threshold that is given on thein metric unit: gram (g) (Suppl Fig. 1). Determination of the environmental noise generated by the device The measurement of the environmental noise evoked by the device was performed using a Brüel&Kjear Photon + 4-channel data acquisition system with a Brüel&Kjear 4944B ultrasonic microphone. Before the measurements, all the sensors were calibrated, in addition the ultrasound microphone for which a Larson and Davies CAL200 Class 1 calibrator was used. The following instrument settings were applied: sampling frequency: 192,000 Hz (evaluable frequency: 85 kHz), measurement duration: variable between 10 and 20 s, depending on operating conditions. To perform the measurement, the microphone was placed inside the device, in the middle of the test compartment, secured to let it hang vertically downward. The evaluations and diagrams were produced by using RT Pro Photon 7.41 software, which can be connected directly to the data acquisition system, and Audacity software. Animals In the second part of the study, we have validated the newly constructed instrument on male and female white-colored NMRI mice (12–16 weeks, 30–40 g), black-colored C57Bl/6 mice (12–16 weeks, 20–30 g) and Wistar rats (12–16 weeks, 200–250 g). Mice were bred in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of Pécs, while rats were purchased from Toxi-Coop Ltd., Budapest, Hungary. All animals involved in the study were housed in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of Pécs in a standard pathogen-free, temperature-controlled room (24–25°C) with a 12-hour light-dark cycle. Food and water were provided ad libitum . Ethics Experiments were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1998), to the European Communities Council Directive of 2010/63/EU and to the requirements of the International Association for the Study of Pain (IASP). Experiments were approved by the Ethics Committee on Animal Research of University of Pécs (license numbers: BA02/2000-71/2022). Animal studies are reported in compliance with the ARRIVE guidelines [ 21 ]. All efforts were made to minimize animal suffering and to reduce the number of animals used. Before the experiments, animals were habituated to the measurement conditions, and those results were not used in the final calculation. Animals were monitored at least once daily for general health and signs of pain or distress, and more frequently in the immediate postoperative period. Postoperative analgesics were not administered because they would interfere with the development and assessment of mechanical hypersensitivity in these models; animals were therefore closely monitored, and no unexpected adverse events were observed. Humane endpoints such as persistent severe distress or > 20% body weight loss were predefined but were not reached in any animal. Touch sensitivity measurements with dynamic plantar aestesiometer (DPA) Validation of the results gained with ADPA was performed by comparing them with those obtained with the long-established manual DPA device (Ugo Basile, Comerio, Italy). Animals were placed into plastic boxes resting on a metal mesh. After a 10-minute acclimatization period, the threshold was measured by increasing force on the plantar surface with a small diameter unsharpened metal needle (max. force: 10 g, ramp: 4 s in mice and max. force: 50 g, ramp: 4 s in rats). The electronic unit recorded the force at which the animals withdrew their paws due to pain, referred to as the mechanonociceptive threshold [ 7 ]. Three values were assessed and averaged on both hindpaws. Assessment of the mechanonociceptive threshold of the hindpaw with ADPA: baseline measurements Baseline mechanonociceptive threshold of the hindpaw was determined using ADPA, which exposed the hindlimb of the experimental animal to mechanical stimuli reaching the pain threshold similarly to the conventional DPA method. The animals were allowed to move freely on a raised metal grid in separate compartments of the device. During the acclimatisation and during the measurement, the camera system in the device continuously detected the position and posture of the animal. The digital motion picture recorded during the video observation was analysed in real time by the computer software. At the appropriate moment - when the animal was at rest -, the actuator unit of the device precisely positioned the blunt-end steel needle - based on the defined coordinates - and stimulated the middle part of the animal's hindpaw. As the steel needle rised, it applied progressively increasing pressure to the plantar surface of the hindpaw, until the animal showed defensive reaction: the lifting of the hindlimb. When this paw withdrawal response developed, the pain threshold value occurred on the monitor of the computer connected to the apparatus. The average value of the mechanonociceptive threshold was calculated from the results of two or three different measurements. Modelling acute pain: Plantar skin-muscle incision One of the most widely used acute postoperative pain models is the plantar skin and muscle incision model, which is associated with typical mechanical tissue injury as a sign of a significant reduction in the mechanonociceptive threshold. The resultant mechanical hyperalgesia is most pronounced on the first day after incision and is observed for approximately 2–3 days [ 22 , 23 ]. Postoperative pain was induced by an incision of the right hindpaw of the mice or rats on day 0 of the experiment with ketamine (Richter Gedeon, Hungary, 100 mg/kg, i.p.)-xylazine (Eurovet Animal Health BV, The Netherlands, 5 mg/kg, i.p.) anaesthesia [ 9 , 22 ]. In the middle of the right hindpaw, an incision was made through the skin, fascia, and the plantar flexor digitorum brevis muscle, the length of which was 0.5 cm and 1 cm in mice and rats, respectively. The wound was closed with sterile 4.0 silk thread and treated topically with povidone-iodine. The non-operated hindpaws were served as controls. The mechanonociceptive thresholds were assessed with both ADPA and manual DPA before (baseline measurements) and 24 h after surgery (Fig. 2 A). Modelling chronic pain: Partial sciatic nerve ligation (PSNL) Partial sciatic nerve ligation (PSNL) is a reliable and widely used disease model of traumatic neuropathic pain in rodents. Surgery results in significant damage to thinly myelinated and unmyelinated fibres, leading to abnormal sensory functions such as hyperalgesia, without paralysis of motor functions [ 24 ]. The resulting lesions persist from day 3 to day 21 after surgery [ 24 , 25 ]. The surgical procedure was performed under ketamine-xylazine anaesthesia (100 mg/kg − 5 mg/kg i.p.). Partial nerve ligation was performed using 8.0 (mice) and 6.0 (rats) silk surgical thread by tying a tight knot around approximately 1/3 − 1/2 of the diameter of the sciatic nerve [ 11 , 24 , 26 ]. The skin was secured with interrupted stitches using 4.0 suture thread and the wound site was disinfected with povidone-iodine solution at the end of surgery. Mechanonociceptive threshold measurements were performed before (baseline measurement) and 7 days after PSNL (Fig. 2 B). Experimental design, randomisation and blinding For all experiments, the primary outcome measure was the mechanonociceptive threshold of the hindpaw, expressed in grams. Each animal served as its own control, with mechanonociceptive thresholds obtained using both manual DPA and ADPA and compared between the injured and contralateral hindpaw and between baseline and post‑surgical time points. Consequently, no separate randomisation to independent treatment and control groups was performed. Blinding of the experimenter to the surgical status of the hindpaw and the time point was not feasible because the incision and sutures were clearly visible during behavioural testing, which may introduce some risk of bias in outcome assessment. No a priori inclusion or exclusion criteria were defined. All animals that entered the experiments completed the study and were included in the final analysis; no data points were excluded. Statistical analysis Experimental data were evaluated with GraphPad Prism 8 software. Each column of the graphs represents the mean ± SEM of n = 12–19 animals per group for baseline measurements and n = 6 animals per group in the acute and chronic pain models. Data were statistically analysed with one‑way ANOVA followed by Bonferroni post hoc test. Sample sizes were based on our previous studies using similar rodent pain models and behavioural endpoints and were considered sufficient to detect biologically relevant differences; no formal a priori power calculation was performed. Assumptions of the applied parametric tests were not formally tested but were judged to be reasonable based on the distribution of the data as displayed in GraphPad Prism. Results Environmental noise generated by the ADPA device has low effect on the behavioural results Across the 1–80 kHz frequency domain, the environmental noise was typically within 20–25 dB SPL. The highest auditory sensitivity was observed between 10 and 20 kHz, where the hearing threshold reached its minimum of approximately 10–15 dB SPL. Within this frequency range, the measured noise level (e.g., at 16 kHz ≈ 20 dB SPL) exceeded the threshold by only 5–10 dB, indicating that the sound stimulus was perceptible but of low intensity (Fig. 3 .A). During the auditory brainstem response (ABR) threshold measurements, the evoked noise of the device was smaller than both the mouse and rats hearing spectrum in all examined frequency levels (Fig. 3 .B). These results revealed that most of the measured sound levels remained below the species’ auditory sensitivity range. Baseline mechanonociceptive thresholds on injured and intact hindpaws As a first step, the baseline mechanonociceptive threshold values were determined on the hindpaws of the animals measured with both DPA and ADPA devices. The results obtained were consistent across the different animals (Fig. 4 . A-C). The average of the 3 consecutive measurements was 8.40 ± 0.20 g and 8.23 ± 0.11 g in the NMRI, 8.29 ± 0.14 g and 8.36 ± 0.16 g in C57/Bl6 mice measured with DPA on right and left side, respectively (Fig. 4 . A-B). Results measured with ADPA (8.32 ± 0.17 g, 7.97 ± 0.12 g in the NMRI and 7.91 ± 0.13 g, 7.89 ± 0.16 g in the C57/BL6 mice on right and left side, respectively) were not statistically significant from the DPA results, demonstrated the proper functioning of the ADPA. The mechanonociceptive threshold results obtained from Wistar rats were also statistically not different measured by DPA (43.83 ± 0.36 g and 43.91 ± 0.50 g) and ADPA (44.75 ± 0.98 g and 45.57 ± 0.71 g) on right and left side, respectively (Fig. 4 /C). Measurements of acute pain In mice (Fig. 5 . A-B), the baseline thresholds before the surgery were on average between 8–9 grams measured with both devices. Following the skin–muscle incision, thresholds significantly decreased by approximately 50–60% on both day 1 and day 2, with values dropping to an average of 4 grams. Similarly, in the rats (Fig. 5 . C-D), baseline thresholds were 45–50 grams, which significantly decreased by 40–50% on post-operative days 1 and 2, reaching an average of 25–30 grams. No significant differences were observed in the results measured with the two different devices. Measurements of chronic pain In mice (Fig. 6 . A), the average of the baseline thresholds before the surgery were on between 8–9 grams measured with both devices. The sciatic nerve ligation induced a robust, statistically significant drop of the mechanonociceptive thresholds with no difference in the results measured by the different devices. Seven days after the surgery, the percentage change of the initial control threshold values were − 37.85 + 3.12% and − 41.88 + 2.66% measured by DPA and ADPA, respectively (Fig. 6 .B). In the rats (Fig. 6 . C), baseline thresholds of 42 grams significantly decreased on day 7 reaching a range of 20–30 grams for both measurements. No significant differences were found between the percentage changes of the results (-42.95 + 4.38% and − 47.25 + 4.37% on day 7) measured by the DPA or ADPA, respectively (Fig. 6 .D). Discussion and Conclusion In the present study, we have demonstrated the development and validation of a novel high-precision automated device that applies computer-controlled mechanical stimulus as well as parallel detection and digital analysis of the nocifensive reaction in rodents, therefore providing an objective assessment of mechanical pain threshold values. The accuracy of our ADPA equipment was validated by correlating the results with those obtained by manual testing with conventional DPA. The automated measurement of the mechanonociceptive threshold can facilitate the in vivo preclinical testing of potential analgesic agents and help to obtain more objective and reliable results in these studies. The therapeutic success of currently available drugs (opioids, NSAIDs, adjuvant analgesics) for the alleviation of chronic pain is limited; they are often either ineffective, or their long-term use is restricted by severe side effects [ 27 ]. There have been no major breakthroughs in the field of analgesics in recent decades [ 28 ], mainly due to the lack of precise, complex investigational techniques and the translationally relevant animal models as well as the well-designed clinical trials [ 29 ]. Therefore, the constant improvement of all these factors is elevitable in understanding the neuroinflammatory mechanisms involved in pain sensation and identifying those novel therapeutic targets which can be the basis of an innovative therapy. The fact, that the failure of potential analgesics in the clinical trials has been caused partly because of the poor pain measurement techniques, is a well-known problem of the last decades [ 29 , 30 ]. The reason behind this can be explained mainly by suboptimal pain assessment tools, which are often based on simple reflex-based measurements rather than capturing the more complex sensory and emotional dimensions of human pain. As a result, promising drug candidates (e.g. the NK 1 receptor antagonists) that were successfully tested in preclinical models, often failed to prove effectiveness in clinical trials [ 31 , 32 ]. In addition, small numbered or poorly stratified clinical trials have further limited the ability to validate novel therapeutic approaches, which has slowed the pace of significant advances in pain management overall [ 30 ] In recent years, active development has begun in the field of devices used in preclinical pain research. These innovations, which utilize and combine the recent advances in technology and computer processing, target a more precise assessment of pain [ 33 ]. These devices effectively integrate high-speed videography, machine learning, and 3D pose analysis to reveal stimulus-evoked and spontaneous pain-related behaviors in freely moving animals. Several novel innovations have recently appeared on the market, such as specific software providing automated analysis of the rodent’s paw movement [ 34 ], as well as its facial expression according to the grimace scale [ 35 , 36 ], or similar investigation of the behavioural changes that may occur as a result of pain sensation [ 37 , 38 ]. Pain perception is a complex pathophysiological process involving not only the pain pathway but also other areas of the central nervous system responsible for emotional processing [ 39 ], suggesting the importance of studying the animal’s behavioural pain response. However, in case of these methods, the definite paw withdrawal threshold cannot be determined, and the objective quantification of the pain level and severity remains still challenging. Interestingly, an automated equipment has been demonstrated primarily applying optogenetic stimulation for inducing pain, using 100-ms-long pulses of blue light as the pain-inducing photostimulus. As further opportunities, thermal stimulation is also possible evoked by radiant heat (infrared laser light), while mechanical stimulation can be elicited by an indenter tip [ 40 , 41 ]. However, this equipment was validated only for mice, but not for rats, although the application of the latter rodent species is highly essential in the development of novel analgesics. Moreover, the main measurable parameter of this device was the paw withdrawal latency, that is widely used in the assessment of thermal allodynia, but in case of mechanical hyperalgesia it is much more appropriate to determine the paw withdrawal threshold [ 42 – 44 ]. Furthermore, using heat stimuli, the cut-off time should be set carefully, since the prolonged heat exposure can cause greater damage to surrounding tissues and consequent secondary hyperalgesia, strongly limiting the repetition of the latency measurements. Our novel ADPA system was validated in both mice and rats with various fur colors, which feature can substantially influence detection accuracy and complicate automated measurement processes. According to the literature, the average baseline mechanical pain threshold of C57Bl/6 and NMRI mice measured with conventional and electronic von Frey technique varies between 6–10 g [ 45 , 46 ]. Besides, the average baseline mechanonociceptive threshold of Sprague-Dawley and Wistar rats assessed with these methods usually ranges between 40–60 g [ 47 , 48 ]. The mechanonociceptive threshold values assessed with ADPA were highly consistent with those gained with DPA, since no significant differences could be observed between them. This consistency was retained across all experimental set-ups, independently from the investigated species, strain, pathophysiological or physiological condition, fur color and laterality, indicating that the automated system is a valid and reliable tool for assessing mechanonociceptive thresholds in animal pain models. The main advantage of the high precision ADPA, developed in collaboration with Z-Elektronika Ltd., Pécs, Hungary is that it automatically stimulates the animal's hindlimb without human intervention in a computer-controlled manner, thus eliminating the subjective human assessment of the aversive response and the results obtained are independent of the person performing the experiment. Automation (1) increases the objectivity of the tests; (2) reduces the number of tests required due to the objectivity of the tests, which helps to reduce the number of animals used; (3) allows better quality test results to be obtained, (4) increases the comparability and reproducibility of research results. These findings demonstrate that the ADPA device can accurately reproduce manual DPA measurements while providing enhanced efficiency, objectivity, and measurement consistency. It is important to notice, that some environmental noise was detected around 10–20 Hz, where the sound pressure level was only low intensity (10–15 dB) during the measurements. However, this frequency range is in the infrasonic range, whereas mice and rats are sensitive mostly to the ultrasonic range (between 1 kHz – 100 kHz and 250 Hz – 80 kHz, respectively) [ 49 – 51 ], therefore it is unlikely to induce auditory discomfort or physiological stress responses for the animals or to influence the behavioural outcomes. This is further strengthened by the results of the auditory brainstem response measurement, where we did not find any specific values influencing the rodent’s auditory system. It should also be noted that, while the ADPA system provides higher precision and objectivity over conventional manual DPA, it requires longer measurement durations and limits the feasibility of rapid, repeated assessments within short time intervals due to integrated stabilization and multi-step verification protocols that minimize errors from mechanical drift or environmental noise. This trade-off prioritizes data reliability and reproducibility—key for preclinical validation—without compromising overall study outcomes or animal welfare. Future software enhancements could further optimize throughput while retaining these advantages. However, the comparison of the results gained with our novel ADPA device and with the conventional DPA measurements confirmed the reliability of the automated system. We have demonstrated that the newly developed ADPA equipment can be reliably assess the mechanonociceptive threshold, and the obtained results show a strong correlation with those derived from the conventional manual DPA method. These findings indicate that our device provides a valid and reproducible alternative to existing approaches, while reducing operator-dependent variability. Advancing translationally relevant research requires continuous development of novel standardized approaches to increase measurement accuracy, inter-study comparability, and the translation of preclinical pain sensitivity into human therapeutic contexts. Declarations Acknowledgements The authors are grateful to Boglárka Ács to her expert technical assistance. Funding information This study was supported by 2020-1.1.2-PIACI-KFI-2021-00255, by the Hungarian Research Network (HUN-REN, Chronic Pain Research Group, Pécs) and the National Brain Research Program 3.0 (NAP 3.0), as well as by the Hungarian National Research, Development and Innovation Office (OTKA K134214, NKFIH FK 146283). Project no. TKP2021-EGA-13has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the EGA-13 funding scheme. Project no. RRF-2.3.1-21-2022-00015 has been implemented with the support provided by the European Union. CRediT authorship contribution statement Dima Fayiz Barakat Alsou’b: Investigation; Methodology, Eszter Kepe: Investigation; Methodology, Zsófia Hajna: Project administration, Supervision, Writing - original draft Gyula Ulrich: Conceptualization, Funding acquisition, Resources, Erika Pintér: Conceptualization, Funding acquisition, Resources, Supervision, Writing - review & editing Valéria Tékus: Conceptualization, Project administration, Supervision, Writing - original draft, review & editing Data availability The datasets underlying the figures are embedded in the GraphPad Prism files used to generate the graphs and can be accessed by clicking on the individual data points; these files are available from the corresponding author on reasonable request. Additional information Gy. U. is an employee at Z-electronic Ltd. who had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results. E.P is shareholder and V.T. is an employee of PharmInVivo Ltd. The other 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. References Raja, S. N. et al. The revised International Association for the Study of Pain definition of pain: concepts, challenges, and compromises. Pain 161 , 1976–1982. https://doi.org/10.1097/j.pain.0000000000001939 (2020). Smith, A. F., Plumb, A. N., Berardi, G. & Sluka, K. A. Sex differences in the transition to chronic pain. J. Clin. Invest. 2025;135. https://doi.org/10.1172/JCI191931 Sluka, K. A., Sowers, L. P., Fairbanks, C. A. & Lascelles, B. D. X. Moving beyond measures of pain intensity in preclinical models. 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Deuis, J. R., Dvorakova, L. S. & Vetter, I. Methods Used to Evaluate Pain Behaviors in Rodents. Front. Mol. Neurosci. 2017;10. https://doi.org/10.3389/fnmol.2017.00284 Pitcher, G. M., Ritchie, J. & Henry, J. L. Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands. J. Neurosci. Methods . 87 , 185–193. https://doi.org/10.1016/S0165-0270(99)00004-7 (1999). Reitz, M-C., Hrncic, D., Treede, R-D. & Caspani, O. A comparative behavioural study of mechanical hypersensitivity in 2 pain models in rats and humans. Pain 157 , 1248–1258. https://doi.org/10.1097/j.pain.0000000000000515 (2016). Heffner, H. E. & Heffner, R. S. Hearing ranges of laboratory animals. J. Am. Assoc. Lab. Anim. Sci. 46 , 20–22 (2007). Reynolds, R. P., Kinard, W. L., Degraff, J. J., Leverage, N. & Norton, J. N. Noise in a laboratory animal facility from the human and mouse perspectives. J. Am. Assoc. Lab. Anim. Sci. 49 , 592–597 (2010). Escabi, C. D., Frye, M. D., Trevino, M. & Lobarinas, E. The rat animal model for noise-induced hearing loss. J. Acoust. Soc. Am. 146 , 3692–3709. https://doi.org/10.1121/1.5132553 (2019). Additional Declarations Competing interest reported. Gy. U. is an employee at Z-electronic Ltd. who had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results. E.P is shareholder and V.T. is an employee of PharmInVivo Ltd. The other 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. Supplementary Files SupplementarymaterialSciRep20260313.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 28 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers invited by journal 29 Mar, 2026 Editor invited by journal 18 Mar, 2026 Editor assigned by journal 14 Mar, 2026 Submission checks completed at journal 14 Mar, 2026 First submitted to journal 13 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9116474","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":615371811,"identity":"2274a9fc-9978-442a-8744-9cbf2d406b6f","order_by":0,"name":"Dima Fayiz Barakat Alsou’b","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Dima","middleName":"Fayiz Barakat","lastName":"Alsou’b","suffix":""},{"id":615371813,"identity":"57c05f77-6d65-490d-badc-8ee296290d46","order_by":1,"name":"Eszter Kepe","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Eszter","middleName":"","lastName":"Kepe","suffix":""},{"id":615371816,"identity":"5f682d92-e2de-49bd-b863-52c8f6e69de2","order_by":2,"name":"Zsófia Hajna","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Zsófia","middleName":"","lastName":"Hajna","suffix":""},{"id":615371817,"identity":"addd3fee-b222-411b-83bf-2f1e835f6a40","order_by":3,"name":"Gyula Ulrich","email":"","orcid":"","institution":"Z-Elektronika Ltd","correspondingAuthor":false,"prefix":"","firstName":"Gyula","middleName":"","lastName":"Ulrich","suffix":""},{"id":615371820,"identity":"cb675779-45f2-49fc-b40c-bdeae0a54038","order_by":4,"name":"Erika Pintér","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"","lastName":"Pintér","suffix":""},{"id":615371821,"identity":"94e85a5d-220b-4af1-afda-cd0715b71a02","order_by":5,"name":"Valéria Tékus","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3OsWrDMBCA4TMGZzknq4Sp+woyHgvpswhDunY0pBAFg7I0ZG1pyWNkThBIi1+g0KGh0K7pFkOG2nFKJzkdO+gHwXHoQwJwuf5lvgBgzRDUJwcg7XrdQbyW4JGUR9KszhE4EU/+gTCzmb7nt0O47skRrZbDmIrQvO3glQsbKXmRlCwDRK2jcJWlEfT59AE+rIQKLqlgPiC5kZG38vkSMCkQlJ0strNKsAng5aek1fOkJYcOMiBceoKp+pVAk1Ao/tQQ6CTbov6YQSxH2RVqk9Kizx/vmUptJBhkmy9xGMe9mU5e9nfjmJj5erfP1YWN/IS/o9/M7Mx9O3e5XC5X3TeAlVOaixbkgQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Pécs","correspondingAuthor":true,"prefix":"","firstName":"Valéria","middleName":"","lastName":"Tékus","suffix":""}],"badges":[],"createdAt":"2026-03-13 15:53:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9116474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9116474/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105953244,"identity":"570f4559-0316-4aef-a956-698815959bae","added_by":"auto","created_at":"2026-04-01 19:13:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":301291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-precision automated dynamic plantar aesthesiometer (ADPA) developed by Z-elektronika Ltd. Hungary.\u003c/strong\u003e (A) The device setup used for assessing mechanical sensitivity in rodents. (B) Top-view from the built-in camera, showing 12 individual chambers that allow testing multiple animals simultaneously. (C) Bottom-view from the real-time camera, showing the positioning of the needle in relation to the animal’s hind paw during the measurement.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/7821af35d10e56d6b900dd2d.jpg"},{"id":106093777,"identity":"362d61b5-ae7e-42ce-bd3f-36a386c855f4","added_by":"auto","created_at":"2026-04-03 11:39:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":827314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart summarizing the main procedures of the two surgical intervention models. \u003c/strong\u003e(A) Acute postoperative pain model elicited by plantar skin-muscle incision performed in mice and rats, (B) chronic neuropathic pain model elicited by partial sciatic nerve ligation (PSNL; Seltzer method), performed in mice and rats.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/8fe44677aabfdb19682291a9.jpg"},{"id":105953247,"identity":"0fc54747-9be3-4c9e-82b7-c760010b9e49","added_by":"auto","created_at":"2026-04-01 19:13:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermination of the environmental noise generated by the ADPA device, (A) behavioural threshold, (B) auditory brainstem response (ABR)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/04ce6cb608eefb4c32650c9b.png"},{"id":105953250,"identity":"042463cb-59da-43e9-8a2f-252130fe719b","added_by":"auto","created_at":"2026-04-01 19:13:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBaseline mechanonociceptive threshold measurements obtained using manual (green) and automated (red) dynamic plantar aesthesiometry (DPA) on the right and left hind paws.\u003c/strong\u003e (A) NMRI mice. (B) C57BL/6 mice. (C) Wistar rats. Thresholds are expressed in grams (n = 12-19/group, one-way ANOVA with multiple comparisons; p \u0026lt; 0.05, p \u0026lt; 0.01, *p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/a17452c72fe6224d991aa5fe.png"},{"id":105953248,"identity":"070131b6-ebdb-4a91-83ae-85d01495c59b","added_by":"auto","created_at":"2026-04-01 19:13:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTouch sensitivity measurements using manual (DPA, green) and automated (ADPA, red) dynamic plantar aesthesiometry in acute pain (plantar skin and muscle incision) model.\u003c/strong\u003e The changes of the mechanonociceptive threshold of the mice (A–B) and rats (C–D) assessed before surgery and on post-operative days 1 and 2. (A) and (C) panels represent the mechanonociceptive threshold values in g, while (B) and (D) panels show the percentage change of the initial control values of the injured hindpaws. Data are presented as the mean ± SEM (n=6-6), animals with individual plots (n = 6/group, one-way ANOVA with multiple comparisons; **\u003cstrong\u003ep\u003c/strong\u003e \u0026lt; 0.01, ***\u003cstrong\u003ep\u003c/strong\u003e \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. baseline controls).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/b8183bd0de0a2885d0504b51.png"},{"id":106959772,"identity":"f7655eed-0808-43c6-a984-ed69eaa77f21","added_by":"auto","created_at":"2026-04-15 09:14:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":28651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTouch sensitivity measurements using manual (DPA, green) and automated (ADPA, red) dynamic plantar aesthesiometry in chronic pain (sciatic nerve ligation mononeuropathy) model.\u003c/strong\u003e The changes of the mechanonociceptive threshold of the mice (A–B) and rats (C–D) assessed before surgery and on post-operative day 7. (A) and (C) panels represent the mechanonociceptive threshold values in g, while (B) and (D) panels show the percentage change of the initial control values of the injured hindpaws. Data are presented as the mean ± SEM (n=6-6), animals with individual plots (n = 6/group, one-way ANOVA with multiple comparisons; **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. baseline controls).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/153e761f980a7c30aa9acb37.png"},{"id":106994159,"identity":"96711b14-4e35-421c-b66b-1683f1251a19","added_by":"auto","created_at":"2026-04-15 15:05:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2506590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/f6ce44a7-115a-4889-8ed7-53a1eaf27485.pdf"},{"id":106093183,"identity":"a4313fc1-de13-4ff2-92a5-443cf27dc14b","added_by":"auto","created_at":"2026-04-03 11:35:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":370880,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialSciRep20260313.docx","url":"https://assets-eu.researchsquare.com/files/rs-9116474/v1/9cd2db01b0329913a55c9870.docx"}],"financialInterests":"Competing interest reported. Gy. U. is an employee at Z-electronic Ltd. who had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results. E.P is shareholder and V.T. is an employee of PharmInVivo Ltd. The other 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.","formattedTitle":"Development of high-precision automated dynamic plantar aesthesiometer (ADPA): a promising tool in pain research","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs the International Association for the Study of Pain (IASP) defined, pain is an unpleasant sensory and emotional experience linked to actual or potential tissue damage that affects individuals all over the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Acute pain is adaptive and serves a protective function by signalling potential or avoiding actual tissue damage. If pain persists for weeks to months, the initial acute pain becomes chronic, loses its protective function and becomes maladaptive [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Approximately 20% of the population suffer from chronic pain, with 8% reporting a negative impact on daily life [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Chronic pain associated with neuroplastic changes in the nervous system, induces significant impairment in quality of life and increased healthcare cost as well as huge socio-economic burden for the government of the countries [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePain and its impact vary among individuals and are influenced by environmental, cultural, and lifestyle factors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since animals could not communicate the pain verbally; only the nocifensive reaction (spinal reflexes) evoked by painful stimuli can be measured. Development of a sensitive, objective equipment which can be detect any signs of pain together with the nociceptive responses is challenging. In preclinical animal studies, the most frequently used tools work by measuring the rodents\u0026rsquo;s nocifensive movements (paw withdrawal; shaking, lifting or licking of the affected paws). The harmful stimulus can be either mechanical (Dynamic Plantar Aestesiometer (DPA), Ugo Basile, Italy) [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]; Von Frey filament [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and the Randall-Selitto analgesimeter [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], or heat using increasing/decreasing temperature hot/cold plate or water bath [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Hargreaves apparatus [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Beside these challenging factors, the currently available methods have several other limitations, e.g. most of them are manually operated, moreover, some of them require that the animals should be held in the examiners hand throughout the whole examination time, which is highly stressful for the animals. Even changing the person of the examiner during the experiment, the unfamiliar smell or different mood status can be also stressful for the animals. This stress alone can modify the measurement outcomes, so the results obtained contain many subjective elements and depend heavily on the experience of the examiner, and even result in low standardization, which can reduce the validity of the data and lead to incorrect conclusions. In addition, this subjectivity can reduce the validity of the measured data and lead to false conclusions.\u003c/p\u003e \u003cp\u003eTherefore, the development of pain measurement tools is crucial for collecting objective, reliable data, measuring effectively the pain intensity, detecting the efficacy and dosages of the novel therapeutic agents. Improvement of the pain detecting device helps to minimalize the numbers and the suffering of the animals used in preclinical studies and can elevate the translational value of this preclinical research further increasing the successfulness of analgesic drug research.\u003c/p\u003e \u003cp\u003eBased on this, our experiments aimed to develop a new-generation pain threshold assessment device that automates both the delivery of mechanical stimuli and the evaluation of nocifensive behaviour, thereby reducing human bias and improving the reproducibility and reliability of pain assessment. Furthermore, we also aimed to validate our newly developed equipment in preclinical animal models of acute and chronic pain, for which the determination of mechanical pain threshold and mechanical hyperalgesia/allodynia is essential, and to compare these data to those gained with conventional DPA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment process of the high-precision automated dynamic plantar aesthesiometer (ADPA)\u003c/h2\u003e \u003cp\u003eIn the first part of the project, in collaboration with Z-Elektronika Ltd. we have constructed a new generation mechanical pain threshold measurement apparatus in which the mechanical stimulation of the hindpaw as well as the detection and analysis of the nocifensive behaviour is driven automatically by the computer connected to the equipment. This high-precision automated dynamic plantar aesthesiometer (ADPA) consists of three separated well-ventilated and well-illuminated plastic compartments placed on a raised metal grid. The size of these compartments is 20 cm x 20 cm and allows the parallel measurement of three rats. Moreover, each compartment can be further divided into four parts, and the 10 cm x 10 cm parts allow to measure 12 mice altogether, so the equipment is suitable for the investigation of both rodent strains that are routinely applied in preclinical studies. The equipment consists of 4 cameras, three of which serve the detection of the position and posture of the animal from above, in order to ensure the precise moving of the needle, while the fourth camera is built in the actuator unit and it is responsible for the videorecording of the nocifensive behaviour from below. The linear actuator unit is found below the metal grid, including the blunt-end needle exerting the mechanical stimulation of the hindpaw. The moving of the linear actuator unit and the elevation of the needle is controlled by the system\u0026rsquo;s software based on the videorecorded alignment of the animal. At the bottom of the equipment, a removable tray is found enabling easy and fast cleaning of the device, moreover, the metal grid is exchangeable allowing fast changeover between the investigation of different strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe apparatus is connected to a computer, that runs a user-friendly, artificial intelligence (AI)-driven software controlling the movement of the actuator unit and the needle, as well as performing the video analysis of the nocifensive behaviour. Before starting the measurement, the investigator can choose from normal, control and manual modes, as well as perform several settings, including the strain, sex, treatment and individual identifier of the animal, the minimum body length, the target leg (left or right), the maximum target force, the minimum force (that is only applicable for control measurements), the force time, the number of measurements to be taken per hindlimb, the animal\u0026rsquo;s relax time, the time between measurements, as well as the waiting time after start or the last stop. The device provides the results of the measurements in an individual data table for each animal including the actual maximum measured force, the target maximum force and the measurement number, alongside the video recordings of the measurement from the top and bottom cameras (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e/B-C). These settings allow the automated, real-time measurement of mechanonociceptive threshold that is given on thein metric unit: gram (g) (Suppl Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of the environmental noise generated by the device\u003c/h3\u003e\n\u003cp\u003eThe measurement of the environmental noise evoked by the device was performed using a Br\u0026uuml;el\u0026amp;Kjear Photon\u0026thinsp;+\u0026thinsp;4-channel data acquisition system with a Br\u0026uuml;el\u0026amp;Kjear 4944B ultrasonic microphone. Before the measurements, all the sensors were calibrated, in addition the ultrasound microphone for which a Larson and Davies CAL200 Class 1 calibrator was used. The following instrument settings were applied: sampling frequency: 192,000 Hz (evaluable frequency: 85 kHz), measurement duration: variable between 10 and 20 s, depending on operating conditions. To perform the measurement, the microphone was placed inside the device, in the middle of the test compartment, secured to let it hang vertically downward. The evaluations and diagrams were produced by using RT Pro Photon 7.41 software, which can be connected directly to the data acquisition system, and Audacity software.\u003c/p\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eIn the second part of the study, we have validated the newly constructed instrument on male and female white-colored NMRI mice (12\u0026ndash;16 weeks, 30\u0026ndash;40 g), black-colored C57Bl/6 mice (12\u0026ndash;16 weeks, 20\u0026ndash;30 g) and Wistar rats (12\u0026ndash;16 weeks, 200\u0026ndash;250 g). Mice were bred in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of P\u0026eacute;cs, while rats were purchased from Toxi-Coop Ltd., Budapest, Hungary. All animals involved in the study were housed in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of P\u0026eacute;cs in a standard pathogen-free, temperature-controlled room (24\u0026ndash;25\u0026deg;C) with a 12-hour light-dark cycle. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eEthics\u003c/h3\u003e\n\u003cp\u003e Experiments were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1998), to the European Communities Council Directive of 2010/63/EU and to the requirements of the International Association for the Study of Pain (IASP). Experiments were approved by the Ethics Committee on Animal Research of University of P\u0026eacute;cs (license numbers: BA02/2000-71/2022). Animal studies are reported in compliance with the ARRIVE guidelines [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. All efforts were made to minimize animal suffering and to reduce the number of animals used. Before the experiments, animals were habituated to the measurement conditions, and those results were not used in the final calculation. Animals were monitored at least once daily for general health and signs of pain or distress, and more frequently in the immediate postoperative period. Postoperative analgesics were not administered because they would interfere with the development and assessment of mechanical hypersensitivity in these models; animals were therefore closely monitored, and no unexpected adverse events were observed. Humane endpoints such as persistent severe distress or \u0026gt;\u0026thinsp;20% body weight loss were predefined but were not reached in any animal.\u003c/p\u003e\n\u003ch3\u003eTouch sensitivity measurements with dynamic plantar aestesiometer (DPA)\u003c/h3\u003e\n\u003cp\u003eValidation of the results gained with ADPA was performed by comparing them with those obtained with the long-established manual DPA device (Ugo Basile, Comerio, Italy). Animals were placed into plastic boxes resting on a metal mesh. After a 10-minute acclimatization period, the threshold was measured by increasing force on the plantar surface with a small diameter unsharpened metal needle (max. force: 10 g, ramp: 4 s in mice and max. force: 50 g, ramp: 4 s in rats). The electronic unit recorded the force at which the animals withdrew their paws due to pain, referred to as the mechanonociceptive threshold [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Three values were assessed and averaged on both hindpaws.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of the mechanonociceptive threshold of the hindpaw with ADPA: baseline measurements\u003c/h2\u003e \u003cp\u003eBaseline mechanonociceptive threshold of the hindpaw was determined using ADPA, which exposed the hindlimb of the experimental animal to mechanical stimuli reaching the pain threshold similarly to the conventional DPA method. The animals were allowed to move freely on a raised metal grid in separate compartments of the device. During the acclimatisation and during the measurement, the camera system in the device continuously detected the position and posture of the animal. The digital motion picture recorded during the video observation was analysed in real time by the computer software. At the appropriate moment - when the animal was at rest -, the actuator unit of the device precisely positioned the blunt-end steel needle - based on the defined coordinates - and stimulated the middle part of the animal's hindpaw. As the steel needle rised, it applied progressively increasing pressure to the plantar surface of the hindpaw, until the animal showed defensive reaction: the lifting of the hindlimb. When this paw withdrawal response developed, the pain threshold value occurred on the monitor of the computer connected to the apparatus. The average value of the mechanonociceptive threshold was calculated from the results of two or three different measurements.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModelling acute pain: Plantar skin-muscle incision\u003c/h3\u003e\n\u003cp\u003eOne of the most widely used acute postoperative pain models is the plantar skin and muscle incision model, which is associated with typical mechanical tissue injury as a sign of a significant reduction in the mechanonociceptive threshold. The resultant mechanical hyperalgesia is most pronounced on the first day after incision and is observed for approximately 2\u0026ndash;3 days [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePostoperative pain was induced by an incision of the right hindpaw of the mice or rats on day 0 of the experiment with ketamine (Richter Gedeon, Hungary, 100 mg/kg, i.p.)-xylazine (Eurovet Animal Health BV, The Netherlands, 5 mg/kg, i.p.) anaesthesia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the middle of the right hindpaw, an incision was made through the skin, fascia, and the plantar flexor digitorum brevis muscle, the length of which was 0.5 cm and 1 cm in mice and rats, respectively. The wound was closed with sterile 4.0 silk thread and treated topically with povidone-iodine. The non-operated hindpaws were served as controls. The mechanonociceptive thresholds were assessed with both ADPA and manual DPA before (baseline measurements) and 24 h after surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eModelling chronic pain: Partial sciatic nerve ligation (PSNL)\u003c/h3\u003e\n\u003cp\u003ePartial sciatic nerve ligation (PSNL) is a reliable and widely used disease model of traumatic neuropathic pain in rodents. Surgery results in significant damage to thinly myelinated and unmyelinated fibres, leading to abnormal sensory functions such as hyperalgesia, without paralysis of motor functions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The resulting lesions persist from day 3 to day 21 after surgery [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe surgical procedure was performed under ketamine-xylazine anaesthesia (100 mg/kg\u0026thinsp;\u0026minus;\u0026thinsp;5 mg/kg i.p.). Partial nerve ligation was performed using 8.0 (mice) and 6.0 (rats) silk surgical thread by tying a tight knot around approximately 1/3\u0026thinsp;\u0026minus;\u0026thinsp;1/2 of the diameter of the sciatic nerve [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The skin was secured with interrupted stitches using 4.0 suture thread and the wound site was disinfected with povidone-iodine solution at the end of surgery.\u003c/p\u003e \u003cp\u003eMechanonociceptive threshold measurements were performed before (baseline measurement) and 7 days after PSNL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design, randomisation and blinding\u003c/h2\u003e \u003cp\u003eFor all experiments, the primary outcome measure was the mechanonociceptive threshold of the hindpaw, expressed in grams. Each animal served as its own control, with mechanonociceptive thresholds obtained using both manual DPA and ADPA and compared between the injured and contralateral hindpaw and between baseline and post‑surgical time points. Consequently, no separate randomisation to independent treatment and control groups was performed. Blinding of the experimenter to the surgical status of the hindpaw and the time point was not feasible because the incision and sutures were clearly visible during behavioural testing, which may introduce some risk of bias in outcome assessment. No a priori inclusion or exclusion criteria were defined. All animals that entered the experiments completed the study and were included in the final analysis; no data points were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperimental data were evaluated with GraphPad Prism 8 software. Each column of the graphs represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of n\u0026thinsp;=\u0026thinsp;12\u0026ndash;19 animals per group for baseline measurements and n\u0026thinsp;=\u0026thinsp;6 animals per group in the acute and chronic pain models. Data were statistically analysed with one‑way ANOVA followed by Bonferroni post hoc test. Sample sizes were based on our previous studies using similar rodent pain models and behavioural endpoints and were considered sufficient to detect biologically relevant differences; no formal a priori power calculation was performed. Assumptions of the applied parametric tests were not formally tested but were judged to be reasonable based on the distribution of the data as displayed in GraphPad Prism.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnvironmental noise generated by the ADPA device has low effect on the behavioural results\u003c/h2\u003e \u003cp\u003eAcross the 1\u0026ndash;80 kHz frequency domain, the environmental noise was typically within 20\u0026ndash;25 dB SPL. The highest auditory sensitivity was observed between 10 and 20 kHz, where the hearing threshold reached its minimum of approximately 10\u0026ndash;15 dB SPL. Within this frequency range, the measured noise level (e.g., at 16 kHz\u0026thinsp;\u0026asymp;\u0026thinsp;20 dB SPL) exceeded the threshold by only 5\u0026ndash;10 dB, indicating that the sound stimulus was perceptible but of low intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A).\u003c/p\u003e \u003cp\u003eDuring the auditory brainstem response (ABR) threshold measurements, the evoked noise of the device was smaller than both the mouse and rats hearing spectrum in all examined frequency levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B). These results revealed that most of the measured sound levels remained below the species\u0026rsquo; auditory sensitivity range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBaseline mechanonociceptive thresholds on injured and intact hindpaws\u003c/h2\u003e \u003cp\u003eAs a first step, the baseline mechanonociceptive threshold values were determined on the hindpaws of the animals measured with both DPA and ADPA devices. The results obtained were consistent across the different animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A-C). The average of the 3 consecutive measurements was 8.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 g and 8.23\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.11 g in the NMRI, 8.29\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.14 g and 8.36\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.16 g in C57/Bl6 mice measured with DPA on right and left side, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A-B). Results measured with ADPA (8.32\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.17 g, 7.97\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.12 g in the NMRI and 7.91\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.13 g, 7.89\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.16 g in the C57/BL6 mice on right and left side, respectively) were not statistically significant from the DPA results, demonstrated the proper functioning of the ADPA. The mechanonociceptive threshold results obtained from Wistar rats were also statistically not different measured by DPA (43.83\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.36 g and 43.91\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.50 g) and ADPA (44.75\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.98 g and 45.57\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.71 g) on right and left side, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e/C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of acute pain\u003c/h2\u003e \u003cp\u003eIn mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A-B), the baseline thresholds before the surgery were on average between 8\u0026ndash;9 grams measured with both devices. Following the skin\u0026ndash;muscle incision, thresholds significantly decreased by approximately 50\u0026ndash;60% on both day 1 and day 2, with values dropping to an average of 4 grams. Similarly, in the rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. C-D), baseline thresholds were 45\u0026ndash;50 grams, which significantly decreased by 40\u0026ndash;50% on post-operative days 1 and 2, reaching an average of 25\u0026ndash;30 grams. No significant differences were observed in the results measured with the two different devices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of chronic pain\u003c/h2\u003e \u003cp\u003eIn mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. A), the average of the baseline thresholds before the surgery were on between 8\u0026ndash;9 grams measured with both devices. The sciatic nerve ligation induced a robust, statistically significant drop of the mechanonociceptive thresholds with no difference in the results measured by the different devices. Seven days after the surgery, the percentage change of the initial control threshold values were \u0026minus;\u0026thinsp;37.85\u0026thinsp;+\u0026thinsp;3.12% and \u0026minus;\u0026thinsp;41.88\u0026thinsp;+\u0026thinsp;2.66% measured by DPA and ADPA, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.B).\u003c/p\u003e \u003cp\u003eIn the rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. C), baseline thresholds of 42 grams significantly decreased on day 7 reaching a range of 20\u0026ndash;30 grams for both measurements. No significant differences were found between the percentage changes of the results (-42.95\u0026thinsp;+\u0026thinsp;4.38% and \u0026minus;\u0026thinsp;47.25\u0026thinsp;+\u0026thinsp;4.37% on day 7) measured by the DPA or ADPA, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.D).\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion and Conclusion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003cp\u003eIn the present study, we have demonstrated the development and validation of a novel high-precision automated device that applies computer-controlled mechanical stimulus as well as parallel detection and digital analysis of the nocifensive reaction in rodents, therefore providing an objective assessment of mechanical pain threshold values. The accuracy of our ADPA equipment was validated by correlating the results with those obtained by manual testing with conventional DPA. The automated measurement of the mechanonociceptive threshold can facilitate the \u003cem\u003ein vivo\u003c/em\u003e preclinical testing of potential analgesic agents and help to obtain more objective and reliable results in these studies.\u003c/p\u003e \u003cp\u003eThe therapeutic success of currently available drugs (opioids, NSAIDs, adjuvant analgesics) for the alleviation of chronic pain is limited; they are often either ineffective, or their long-term use is restricted by severe side effects [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. There have been no major breakthroughs in the field of analgesics in recent decades [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], mainly due to the lack of precise, complex investigational techniques and the translationally relevant animal models as well as the well-designed clinical trials [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, the constant improvement of all these factors is elevitable in understanding the neuroinflammatory mechanisms involved in pain sensation and identifying those novel therapeutic targets which can be the basis of an innovative therapy.\u003c/p\u003e \u003cp\u003eThe fact, that the failure of potential analgesics in the clinical trials has been caused partly because of the poor pain measurement techniques, is a well-known problem of the last decades [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The reason behind this can be explained mainly by suboptimal pain assessment tools, which are often based on simple reflex-based measurements rather than capturing the more complex sensory and emotional dimensions of human pain. As a result, promising drug candidates (e.g. the NK\u003csub\u003e1\u003c/sub\u003e receptor antagonists) that were successfully tested in preclinical models, often failed to prove effectiveness in clinical trials [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, small numbered or poorly stratified clinical trials have further limited the ability to validate novel therapeutic approaches, which has slowed the pace of significant advances in pain management overall [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn recent years, active development has begun in the field of devices used in preclinical pain research. These innovations, which utilize and combine the recent advances in technology and computer processing, target a more precise assessment of pain [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These devices effectively integrate high-speed videography, machine learning, and 3D pose analysis to reveal stimulus-evoked and spontaneous pain-related behaviors in freely moving animals.\u003c/p\u003e \u003cp\u003eSeveral novel innovations have recently appeared on the market, such as specific software providing automated analysis of the rodent\u0026rsquo;s paw movement [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], as well as its facial expression according to the grimace scale [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], or similar investigation of the behavioural changes that may occur as a result of pain sensation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Pain perception is a complex pathophysiological process involving not only the pain pathway but also other areas of the central nervous system responsible for emotional processing [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], suggesting the importance of studying the animal\u0026rsquo;s behavioural pain response. However, in case of these methods, the definite paw withdrawal threshold cannot be determined, and the objective quantification of the pain level and severity remains still challenging.\u003c/p\u003e \u003cp\u003eInterestingly, an automated equipment has been demonstrated primarily applying optogenetic stimulation for inducing pain, using 100-ms-long pulses of blue light as the pain-inducing photostimulus. As further opportunities, thermal stimulation is also possible evoked by radiant heat (infrared laser light), while mechanical stimulation can be elicited by an indenter tip [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, this equipment was validated only for mice, but not for rats, although the application of the latter rodent species is highly essential in the development of novel analgesics. Moreover, the main measurable parameter of this device was the paw withdrawal latency, that is widely used in the assessment of thermal allodynia, but in case of mechanical hyperalgesia it is much more appropriate to determine the paw withdrawal threshold [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, using heat stimuli, the cut-off time should be set carefully, since the prolonged heat exposure can cause greater damage to surrounding tissues and consequent secondary hyperalgesia, strongly limiting the repetition of the latency measurements.\u003c/p\u003e \u003cp\u003eOur novel ADPA system was validated in both mice and rats with various fur colors, which feature can substantially influence detection accuracy and complicate automated measurement processes. According to the literature, the average baseline mechanical pain threshold of C57Bl/6 and NMRI mice measured with conventional and electronic von Frey technique varies between 6\u0026ndash;10 g [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Besides, the average baseline mechanonociceptive threshold of Sprague-Dawley and Wistar rats assessed with these methods usually ranges between 40\u0026ndash;60 g [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The mechanonociceptive threshold values assessed with ADPA were highly consistent with those gained with DPA, since no significant differences could be observed between them. This consistency was retained across all experimental set-ups, independently from the investigated species, strain, pathophysiological or physiological condition, fur color and laterality, indicating that the automated system is a valid and reliable tool for assessing mechanonociceptive thresholds in animal pain models.\u003c/p\u003e \u003cp\u003eThe main advantage of the high precision ADPA, developed in collaboration with Z-Elektronika Ltd., P\u0026eacute;cs, Hungary is that it automatically stimulates the animal's hindlimb without human intervention in a computer-controlled manner, thus eliminating the subjective human assessment of the aversive response and the results obtained are independent of the person performing the experiment. Automation (1) increases the objectivity of the tests; (2) reduces the number of tests required due to the objectivity of the tests, which helps to reduce the number of animals used; (3) allows better quality test results to be obtained, (4) increases the comparability and reproducibility of research results. These findings demonstrate that the ADPA device can accurately reproduce manual DPA measurements while providing enhanced efficiency, objectivity, and measurement consistency.\u003c/p\u003e \u003cp\u003eIt is important to notice, that some environmental noise was detected around 10\u0026ndash;20 Hz, where the sound pressure level was only low intensity (10\u0026ndash;15 dB) during the measurements. However, this frequency range is in the infrasonic range, whereas mice and rats are sensitive mostly to the ultrasonic range (between 1 kHz \u0026ndash; 100 kHz and 250 Hz \u0026ndash; 80 kHz, respectively) [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], therefore it is unlikely to induce auditory discomfort or physiological stress responses for the animals or to influence the behavioural outcomes. This is further strengthened by the results of the auditory brainstem response measurement, where we did not find any specific values influencing the rodent\u0026rsquo;s auditory system.\u003c/p\u003e \u003cp\u003eIt should also be noted that, while the ADPA system provides higher precision and objectivity over conventional manual DPA, it requires longer measurement durations and limits the feasibility of rapid, repeated assessments within short time intervals due to integrated stabilization and multi-step verification protocols that minimize errors from mechanical drift or environmental noise. This trade-off prioritizes data reliability and reproducibility\u0026mdash;key for preclinical validation\u0026mdash;without compromising overall study outcomes or animal welfare. Future software enhancements could further optimize throughput while retaining these advantages. However, the comparison of the results gained with our novel ADPA device and with the conventional DPA measurements confirmed the reliability of the automated system.\u003c/p\u003e \u003cp\u003eWe have demonstrated that the newly developed ADPA equipment can be reliably assess the mechanonociceptive threshold, and the obtained results show a strong correlation with those derived from the conventional manual DPA method. These findings indicate that our device provides a valid and reproducible alternative to existing approaches, while reducing operator-dependent variability. Advancing translationally relevant research requires continuous development of novel standardized approaches to increase measurement accuracy, inter-study comparability, and the translation of preclinical pain sensitivity into human therapeutic contexts.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Boglárka Ács to her expert technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by 2020-1.1.2-PIACI-KFI-2021-00255, by the Hungarian Research Network (HUN-REN, Chronic Pain Research Group, Pécs) and the National Brain Research Program 3.0 (NAP 3.0), as well as by the Hungarian National Research, Development and Innovation Office (OTKA K134214, NKFIH FK 146283). Project no. TKP2021-EGA-13has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the\u0026nbsp;EGA-13\u0026nbsp;funding scheme. Project no. RRF-2.3.1-21-2022-00015 has been implemented with the support provided by the European Union.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDima Fayiz Barakat Alsou’b:\u0026nbsp;\u003c/strong\u003eInvestigation; Methodology,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEszter Kepe:\u0026nbsp;\u003c/strong\u003eInvestigation; Methodology,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZsófia Hajna:\u0026nbsp;\u003c/strong\u003eProject administration, Supervision, Writing - original draft\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGyula Ulrich:\u0026nbsp;\u003c/strong\u003eConceptualization, Funding acquisition, Resources,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eErika Pintér:\u0026nbsp;\u003c/strong\u003eConceptualization, Funding acquisition, Resources, Supervision, Writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValéria Tékus:\u0026nbsp;\u003c/strong\u003eConceptualization, Project administration, Supervision, Writing - original draft, review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets underlying the figures are embedded in the GraphPad Prism files used to generate the graphs and can be accessed by clicking on the individual data points; these files are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGy. U. is an employee at Z-electronic Ltd. who had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results. E.P is shareholder and V.T. is an employee of PharmInVivo Ltd. The other 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.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRaja, S. N. et al. 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To develop novel analgesics, \u003cem\u003ein vivo\u003c/em\u003e preclinical assessment of the pain threshold is inevitable. Investigation of the nociception in rodents is still challenging, since most of the currently available methods are manually operated. So, the results highly depend on the experience of the examiner and can be significantly biased by subjective human factors. To improve this translational research paradigm, advanced tools are needed in this field. Therefore, the aim of the present study was to develop a new generation automated pain assessment device.\u003c/p\u003e\n\u003cp\u003eIn collaboration with Z-Elektronika Ltd., Pécs, Hungary we have designed and validated high-precision automated dynamic plantar aesthesiometer (ADPA) that is suitable for the assessment of mechanonociceptive threshold in rats and mice. It utilizes artificial intelligence (AI) to automatically recognize the animals investigated. The system's software controls the mechanical stimulation of the hindpaws with simultaneous video recording of the nocifensive reaction and analysis of the pain thresholds. The main advantage of ADPA is the automated, computer-controlled induction and evaluation of the pain threshold, increasing the quality, comparability, reproducibility, and objectivity of the results. This device may significantly enhance the accuracy of pain assessment in animal models and contribute to improved preclinical pain research.\u003c/p\u003e","manuscriptTitle":"Development of high-precision automated dynamic plantar aesthesiometer (ADPA): a promising tool in pain research","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 19:13:36","doi":"10.21203/rs.3.rs-9116474/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-28T08:36:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T15:49:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T02:11:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25799816294153199896742443622762512658","date":"2026-04-21T21:20:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120486195820420784332431087762206026047","date":"2026-04-17T13:14:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310987491130069715138039061451036469231","date":"2026-03-31T06:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T20:32:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-18T06:18:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-14T11:44:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-14T11:44:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-13T15:42:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09127d5a-58ef-4890-8dd0-387bc7121ded","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":65478172,"name":"Biological sciences/Biological techniques"},{"id":65478173,"name":"Biological sciences/Drug discovery"},{"id":65478174,"name":"Physical sciences/Engineering"},{"id":65478175,"name":"Health sciences/Medical research"},{"id":65478176,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-04-28T08:41:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 19:13:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9116474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9116474","identity":"rs-9116474","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}