The Role of Microcirculatory Dysfunction During Paclitaxel Treatment as a Critical Co-Factor for the Development of Chemotherapy-Induced Peripheral Neuropathy

Abstract

Background

Chemotherapy-induced peripheral neuropathy (CIPN) has a lasting impact on quality of life with a high prevalence and the lack of preventive and causal treatment options. In addition, they are often dose-limiting for curative and palliative oncological therapy. The aim of this study was to systematically investigate the occurrence of paclitaxel-induced peripheral microcirculatory dysfunction and its potential impact on peripheral neuropathy using an experimental in vivo approach.

Methods

77 female 8-week-old mice were randomly assigned into three groups. Each group was exposed to the following intraperitoneal interventions in a blinded fashion: The therapy group was treated with six cycles of paclitaxel. In the control group, mice received six cycles of saline solution. In the vehicle group, animals received six cycles of cremophor. Various microscopic, neurological and biochemical analyses were performed to assess the effects on peripheral nerve function, microcirculation and inflammation.

Results

Von Frey’s neurological test showed a progressive peripheral neuropathy with a significant change in the sensitivity in the sense of hypesthesia of the hind paws in mice treated with paclitaxel. Beside signs of systemic inflammation, intravital microscopic analysis showed a significant reduction in functional capillary density, increased venular leukocyte adherence and endothelial permeability in the paclitaxel-treated mice compared to the control groups. In addition, serological tests and histopathological examinations underlined the paclitaxel-induced inflammation and nerve damage as well as the disturbance of the microcirculation.

Conclusion

The presented findings suggest that paclitaxel-induced microcirculatory disturbances may contribute to the development and severity of CIPN, highlighting the importance of considering microvascular and inflammatory mechanisms in the pathogenesis and management of chemotherapy-induced neuropathy.

Zusammenfassung

Hintergrund

Eine chemotherapieinduzierte periphere Neuropathie (CIPN) kann sich nachhaltig auf die Lebensqualität auswirken und ist mit einer hohen Prävalenz und einem Mangel an vorbeugenden und kausalen Behandlungsmöglichkeiten verbunden. Dazu kommt noch, dass sie nicht selten dosislimitierend ist für kurative und palliative onkologische Therapien. Ziel dieser Studie war die systematische Untersuchung des Auftretens einer Paclitaxel-induzierten peripheren mikrozirkulatorischen Dysfunktion und deren potenzieller Auswirkungen auf die periphere Neuropathie in einem experimentellen In-vivo-Tierversuch.

Methoden

Es wurden 77 weibliche 8 Wochen alte Mäuse in 3 Gruppen randomisiert. Es handelte sich dabei um eine Doppelblindstudie. Jede Gruppe wurde einer der folgenden intraperitonealen Interventionen ausgesetzt: Die Therapiegruppe erhielt 6 Zyklen mit Paclitaxel. Die Mäuse der Kontrollgruppe erhielten 6 Zyklen einer Kochsalzlösung. In der Vehikel-Kontrollgruppe erhielten die Tiere 6 Zyklen mit Cremophor. Verschiedene mikroskopische, neurologische und biochemische Analysen wurden durchgeführt, um die Auswirkungen auf die peripheren Nervenfunktionen, Mikrozirkulation und Inflammation zu evaluieren.

Ergebnisse

Die klinisch-neurologische Sensibilitätsprüfung zeigte eine progressive periphere Neuropathie mit signifikanten Sensibilitätsstörungen (Hypästhesien) der Hinterpfoten bei den mit Paclitaxel behandelten Mäusen. Neben Anzeichen einer systemischen Entzündung fanden intravitale mikroskopische Analysen auch eine signifikante Verringerung der funktionellen Kapillardichte, eine Zunahme der Leukozytenadhäsion in den Venolen und eine erhöhte endotheliale Durchlässigkeit in den mit Paclitaxel behandelten Mäusen verglichen mit den Kontrollgruppen. Auch die serologischen Tests und histopathologischen Untersuchungen wiesen Paclitaxel-induzierte Entzündungen und Nervenschädigungen sowie Störungen der Mikrozirkulation nach.

Schlussfolgerung

Die dargelegten Ergebnisse zeigen, dass Paclitaxel-induzierte mikrozirkulatorische Störungen zur Entwicklung und Schwere einer CIPN beitragen können. Damit wird auch klar, wie wichtig die Beachtung von mikrovaskulären und entzündlichen Mechanismen bei der Pathogenese und dem Management einer chemotherapieinduzierten Neuropathie ist.

Keywords

chemotherapy-induced peripheral neuropathy (CIPN) - paclitaxel - intravital fluorescence microscopy - microvascular damage - endothelial lesions - inflammation

Schlüsselwörter

chemotherapieinduzierte periphere Neuropathie (CIPN) - Paclitaxel - intravitale Fluoreszenzmikroskopie - mikrovaskuläre Schäden - endotheliale Läsion - Inflammation

Introduction

With increasing incidence of cancer and simultaneously higher survival rates, the long-term toxic side effects of oncological therapies, which significantly impair the quality of life, are becoming more and more important [1]. Chemotherapy-induced peripheral neuropathies (CIPNs) are one of the most common neurological side effects of tumor therapy with a prevalence of up to 85% with paclitaxel [2] [3]. CIPNs vary in incidence and prevalence depending on the therapeutic agent [4]. They occur mainly with treatments involving platinum derivatives, vinca alkaloids, taxanes, proteasome inhibitors and immunomodulators, but also with modern antibody-based therapies [5]. They often arise in a temporal and dose-dependent (cumulative) relationship with the application [4] [6] [7] [8].

Currently, there are no agents capable of preventing CIPN. Newer drugs such as duloxetine alone or in combination with pregabalin can only reduce resulting pain, myalgia, and arthralgia [9] [10]. Patients suffer acutely during chemotherapy and then often chronically, mainly due to sensory and motor symptoms, including paresthesia and dysesthesia, heat/cold hyperalgesia and tingling [2] [7] [8] [11] [12]. In addition, painful symptoms such as burning and stabbing (neuropathic) pain may also occur [13] [14]. These manifestations develop in a stocking-and-glove distribution due to the neurotoxic effect preferentially seen on longer axons [15] [16]. The exact localization of the damage on the sensory neuron mainly depends on the chemotherapeutic agent used. Taxanes cause axonal damage of the peripheral nerves leading to changes in morphology, disruption of microtubules [17] and direct damage of mitochondria [18] [19]. Clinical examination documents loss of proprioception and suppression or loss of deep tendon reflexes [13]. Sometimes these symptoms are so severe that they become dose-limiting for the primary tumor therapy [15] [20] [21]. Importantly, all these symptoms can progress into a chronic, life-long course with a lasting impact on the quality of life in up to 23% of all patients [4] [22] [23] [24]. In recent years, numerous chemotherapy-induced neuropathy animal models have been published, most commonly with rodents. These rodent CIPN models are characterized by neurophysiological deficits like those in humans, such as mechanical allodynia, thermal hyper- and hypoalgesia, motor function and morphological changes in DRG and myelinated nerve fibers [25].

Besides these direct neurotoxic mechanisms, treatment with chemotherapeutic agents triggers systemic inflammation. Paclitaxel causes increased production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [18]. This process leads to activation of immune cells with development of neuroinflammation [26]. Numerous syndromes associated with systemic inflammation, such as major surgical trauma, sepsis, or ischemia/reperfusion injury, are associated with microcirculatory dysfunction that may induce, or aggravate, end organ damage [27] [28] [29] [30] [31]. However, despite the high prevalence of CIPN and their significant clinical consequences, the impact of the inflammation-driven microcirculatory dysfunctions on their occurrence and their severity have not been investigated so far.

Thus, the aim of this experimental in vivo study was to systematically investigate the occurrence of paclitaxel-induced peripheral microcirculatory dysfunctions including its consecutive ischemia/reperfusion injury and its potential impact on peripheral neurotoxicity in animal models. To this end, we combined two established robust animal models to represent the microcirculation under induced CIPN.

Materials and Methods

Animal model

All animal experiments were approved by the governmental authority (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, Az. 7221.3–1-016/20) in accordance with the protection of animal act for Germany and the European Directive 2010/63/EU. In total 77 8-week-old homozygous female SKH1-hr hairless mice weighing approximately 25 g were used. They were housed in groups of four to five with a 12 h light-dark cycle under standardized conditions of 21 ± 3 °C and about 60% relative humidity, with steady access to water and food ad libitum. The animals received bedding and building materials for set up of their environment. The hairless mouse ear model was used for the in vivo study of the skin microcirculation [32] [33]. Due to a genetic defect on chromosome 14, the animal loses its fur after postnatal day 10, making the pinna, which we chose as the site of peripheral microcirculation, easily accessible for intravital microscopy of the vessels.

Experimental protocol

The experimental sequence is depicted in [Fig. 1] a. After seven days (day −14 until day −7) of acclimatization to humans by daily handling, training for neurological tests started on day −7. In total 47 mice were randomized to three groups in a blinded manner ([Fig. 1] a). 30 additional mice were randomized into three groups, also blinded, after a 7-day acclimatization period to determine plasma values directly after the last treatment ([Fig. 1] b). All animals were given an identification that did not indicate group affiliation:

Fig. 1 Schematic drawing of the experimental design. a One cohort of animals underwent an acclimatization period in the chronic husbandry (day −14 – day −7), followed by the training phase for neurological tests (day −7 – day −3) and a recovery period until day 0. On day 0 animals received either saline (s), cremophor (c) or paclitaxel (p) for the first time, followed by intravital fluorescence microscopic (IVM) analysis. Treatment with saline (s), cremophor (c) or paclitaxel was repeated 6 times until day 11, with a second IVM analysis. After a recovery period, neurological tests were performed between day 15 and 21. Following euthanasia of animals on day 21, organ and blood samples were harvested. b A second cohort of animals exclusively served for the harvesting of blood and organ samples. For this purpose, animals also underwent an acclimatization period in the chronic husbandry for 7 days (day −7 – day 0), followed by the application of either saline (s), cremophor (c) or paclitaxel (p) on days 0, 2, 4, 7, 9, and 11 with subsequent blood and organ sampling on day 11. Created in BioRender. Reuter, S. (2024) https://BioRender.com/q77m103 [rerif]

Group 1 (Therapy Group)

25 animals received paclitaxel between day 0 and day 11. Animals 1–12 received Taxomedac (Medac GmbH, Wedel, Germany). After this company’s preparation went off the market, we switched to paclitaxel from Kabi (Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) with the same active substance content. The chemotherapeutic agent was injected intraperitoneally at a dose of 5 mg/kg to induce peripheral neuropathy.

Group 2 (Vehicle Group)

26 animals receiving intraperitoneally cremophor in equivalent doses and quantities to the paclitaxel group (macrogol-35-glycerolricinoleate, Cremophor EL from Caesar & Loretz GmbH, Germany and alcohol concentrate 95% from B. Braun AG, Melsungen, Germany) in a mixing ratio of 1 : 1 served as vehicle group.

Group 3 (Control Group)

A further 26 animals with saline served as controls. Cremophor- and saline-treated animals received equivalent volumes accordingly.

All animals were injected 6 times between day 0 and 11. In all groups of the experimental arm A ([Fig. 1] a) intravital fluorescence microscopy (IVM) was performed 60 minutes after the first and the sixth treatment.

Body weight assessment and neurological testing

47 animals ([Fig. 1] a) were weighed twice a week and examined every other day for general and neurological behavior during the 36 days of the entire experimental course. Before application of chemotherapy mice were trained on days −7 to −3 for the von Frey filaments test. After a recovery period of 4 days after the last treatment cycle, neurological testing was performed on days 15, 17 and 21 to assess peripheral neuropathy.

Von Frey filaments test

For the assessment of tactile allodynia, hyper- or hypesthesia the hind limb withdrawal threshold evoked by stimulation of the hind paw with von Frey filaments [34] was determined while mice were placed on a metal mesh floor with 0.6 × 0.6 cm cells. After 10-min accommodation periods mechanical stimuli were applied to both foot pads with five calibrated filaments (Semmes-Weinstein Monofilaments, North Coast Medical, Inc., Morgan Hill, CA, USA) of different elastance ranging from 0.02 g to 2.0 g. Each filament was used for six consecutive stimulations at a frequency of 1/sec. Filaments were applied in ascending order starting with the weakest one. The reaction of the mouse was documented by means of a scoring system (0 = no reaction, 1 = reaction of the mouse by twitching the paw until it was raised slightly and 2 = significant reaction of the mouse with jerky withdrawal of the paw, jumping or changing place). Assuming that all paw withdrawals were brief the paw withdrawal response frequency of all five trials was calculated as percent response frequency according to the following formula: (100/60) × paw withdrawals = % response frequency [34] [35].

During the training week all animals were acclimated to the experimental set-up before any intervention.

The other 30 animals ([Fig. 1] b) were weighed twice a week and examined for general and neurological behavior every other day during the 17 days of the entire experiment, especially on the days of chemotherapy.

A persistent inability to consume sufficient fluids and food, accompanied by a sustained loss > 25 % of initial weight and apathy of the animals, was defined as a potential termination criterion. This criterion did not apply to any animal.

Microscopy

Intravital fluorescence microscopy (IVM)

High resolution multifluorescence microscopy was performed using the Axiotech vario microscope (Carl Zeiss, Jena, Germany) equipped with a 100 W halogen lamp and filter sets for the colors blue (excitation/emission 465–495 nm/> 505 nm), green (510–560 nm/> 575 nm) and ultraviolet (340–380 nm/> 400 nm) using epi-illumination Colibri 7n (Carl Zeiss, Jena, Germany). By use of water-immersion objectives Achroplan ×20/0.50 W and ×63/0.95 W (Carl Zeiss, Jena, Germany) final magnifications of ×200 and ×630 were achieved. Images were recorded at a rate of 30 frames per second by means of the charge-coupled DVD recorder DMR-EX99V (Panasonic, Osaka, Japan). Images were transferred to a DVD system for subsequent off-line analysis using the computer-assisted image analysis system Cap-Image (Dr. Zeintl, Engineering Office, Dreieich, Germany) [36]. Duration of continuous light exposure per observation area was limited to 60 sec to avoid phototoxic effects.

For microcirculatory analysis, animals were anesthetized by a total of 30 µl intraperitoneal injection of 10 % ketamine (100 mg/ml; Betapharm, Vechta, Germany) and 2% xylazine (Rompun 2 %, 20 mg/ml, Bayer AG, Leverkusen, Germany) in a mixing ratio of 1 : 4. Thereafter, retroorbital intravenous injections of 20 µl of 5% fluorescein isothiocyanate-labeled dextran (FITC-dextran, MW 150 kDa, Sigma Aldrich, St. Louis, USA) were performed to enhance the contrast of the microvascular network in the ear ([Fig. 2] a, b). Furthermore 50 µl of 0.5 % rhodamine 6 G (Sigma Aldrich, St. Louis, USA) was injected for staining leukocytes ([Fig. 2] c) and 30 µl of bisbenzimide (Hoechst 33342, 10 µmol/kg) for staining of apoptotic cells. Subsequently, the animals were then placed in lateral position on a 37 °C heated transparent stage. The ear to be investigated was stretched with its ventral surface down on the stage.

Fig. 2 Representative intravital fluorescence microscopic images of the hairless SKH1-hr mice ear displaying a macromolecular leakage, b the capillary network, c postcapillary venules with leukocyte-endothelial cell interaction. Mice were treated with either saline, cremophor or paclitaxel (for further details, please see Materials and Methods). Bars indicate length in µm for a 5 µm, b 50 µm, c 100 µm.

Off-line microcirculatory analysis

Photoprints in low magnification illustrating the areas of interest allowed repetitive assessment of all microcirculatory parameters in identical observation fields along the time. Leukocyte-endothelial cell interaction was assessed in two postcapillary venules as described by Panes et al. and by Vollmar et al. [37] [38]. Next to those vessels, four areas served for analysis of functional capillary density (FCD), ([Fig. 2] b) and number of condensed nuclei. The functional capillary density was defined as the total length of red blood cell-perfused capillaries per observation area and given in [cm/cm²]. Apoptotic cell death was analyzed by counting the number of bisbenzimide-stained cells that showed apoptosis-associated condensation, fragmentation, and crescent-shaped formation of their nuclear chromatin [38] [39]. Flow behavior of leukocytes was analyzed with respect to free floating, rolling, and adherent leukocytes, which were easily identified by their rhodamine 6 G-staining in contrast to non-stained erythrocytes ([Fig. 2] c). Rolling leukocytes were defined as those cells moving along the venular wall at a velocity of less than 40% of that of leukocytes at the centerline and expressed as percentage of the total leukocyte flux. Venular leukocyte adherence was defined as the number of leukocytes not moving or detaching from the endothelial lining of the vessel wall during an observation period of 20 sec and expressed as non-moving cells per endothelial surface [n/mm²]. Thrombocytes, which are also stained by rhodamine 6 G, were differentiated according to their much smaller size compared to leukocytes. Arteriolar and venular diameters were measured via the Cap-Image computer-assisted image analysis system introduced by Klyscz et al. [36]. Microvascular permeability was assessed by venous leakage of FITC-dextran ([Fig. 2] a) and analyzed densitometrically by the ratio of extra- to intravascular fluorescence intensity.

Analysis of blood and organs

Harvesting of blood and organs

At the end of the experiments, animals were exsanguinated in a deep ketamine/xylazine anesthesia, followed by harvesting of blood and tissue samples. We decided not to take individual baseline blood samples prior to start of the protocol due to an expected increase in mortality by such an intervention. The blood samples were centrifuged (3575 rpm for 10 min), and plasma was aliquoted into cryovials and immediately frozen at −20 °C and stored for later analysis. The right front and hind paws were fixed in 4% formalin (Formafix, Grimm med. Logistik GmbH, Torgelow, Germany) and after 48 hours decalcified for at least 4 weeks in Usedecalc (Medite Medical GmbH, Burgdorf, Germany) and later stored embedded in paraffin.

Multiplex analysis

For the assessment of the inflammatory response and damage the following biomarkers in blood were determined in 30 additional mice one hour after last treatment ([Fig. 2] b: day 11) and in 31 previously described animals, who completed the entire protocol (10 of paclitaxel-group, 11 of cremophor-group and 10 of saline-group), ten days after the last treatment cycle ([Fig. 1] a: day 21). All measurements were performed using electrochemiluminescence-based immunoassays (MESO QuickPlex SQ 120, Meso Scale Discovery [MSD], Rockville, MD, USA). Interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1 = CCL2) [40] [41] [42], the chemokines IP-10 (IP-10), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), vascular endothelial growth factor A (VEGF-A), macrophage inflammatory protein 1alpha (MIP-1α = CCL3) [43] [44], matrix-metalloproteinase 9 (MMP-9), brain-derived neurotrophic factor (BDNF) and connecting peptide (C-peptide), [45] were measured using a multiplex U-PLEX assay (MSD, Rockville, MD, USA). The measurement of Neurofilament light chain (NF-L), [46] was performed with the S-PLEX ultrasensitive assay from MSD. All readouts were evaluated using the dedicated software Discovery Workbench 4.0 (MSD, Rockville, MD, USA). For the purpose of statistical analysis, any value below the lowest limit of detection (LLOD) was considered negative and assigned a value of 0 pg/ml in the assay.

Histology and immunohistochemistry

To record morphological differences in peripheral nerves of sciatic nerve and the hind paws with regard to the size of myelin sheaths and axons and their area ratio to each other, we chose morphometry using previously described methods [47] and toluidine blue-nuclear red staining. Immunohistochemical staining with PGP9.5 antibody at a dilution of 1 : 600 (Abcam, Rabbit, Polyclonal, USA) was used to visualize the axons [47]. To determine the axon density within the nerve fibers, the chromogenic reaction was quantified according to the previously published method by Crowe and Yue [48] for the semi-quantitative determination of protein expression. For the immunohistochemical detection of inflammatory tissue reactions, we used the antibody CD11b (ab62817, goat, polyclonal, Abcam, USA) at a dilution of 1 : 500. Anti-acetylated tubulin (Sigma Aldrich, Germany) at a dilution of 1 : 5000 was used for the immunohistochemical examination of microtubule stability [49]. Microscopic images of the semi-thin sections were taken with the microscope BX51 (Olympus, Hamburg, Germany) with 100× oil immersion.

Statistical analysis

The number of animals per group was determined by a power calculation [50]. In case of normal distribution, data are expressed as mean ± SD (standard deviation). In case of failure of normal distribution, data are expressed as median with 25th and 75th percentiles and whiskers indicated minimum and maximum values. Statistical analyses were performed using Graph Pad Prism 8.4.3 (Graph Pad Software, San Diego, USA). Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test, and data of histology and immunohistochemistry were analyzed by one-way ANOVA and Holm-Sidak’s multiple comparisons test. Data were considered significant at p < 0.05. Levels of blood biomarker were assessed, and significances were tested using the Kruskal–Wallis test for independent samples (SPSS 27), followed by pairwise analysis if indicated.

Results

Neurological testing and body weight assessment

Data on the body weight assessment and von Frey filament test are given in [Table 1] and in [Fig. 3]. There was no relevant weight loss at any time during the entire trial ([Table 1]). On day 15 groups did not differ in the hind paw withdrawal threshold ([Fig. 3], left panel). On day 17, the paclitaxel group showed a significantly lower frequency of hind paw withdrawal with the 1.4 g filament compared with the saline and cremophor group ([Fig. 3], middle panel). This effect continued to increase until day 21 ([Fig. 3], right panel).

Table 1 Microcirculatory parameters and body weight in hairless SKH1-hr mice, which were treated with either saline, cremophor or paclitaxel for evaluation of CIPN. Intravital fluorescence microscopy and body weight analysis were performed on day 0 (start of treatment) and on day 11 (end of treatment). Values are given as mean ± SD. Two-way ANOVA and Tukey’s multiple comparisons test revealed no statistically significant differences.

Parameter	Groups	Day 0	Day 11
Arteriolar diameter [µm]	saline	31 ± 3	33 ± 9
	cremophor	31 ± 6	30 ± 6
	paclitaxel	32 ± 4	31 ± 5
Venular diameter [µm]	saline	66 ± 8	66 ± 15
	cremophor	56 ± 12	58 ± 19
	paclitaxel	71 ± 12	64 ± 9
Venular leukocyte flux [n/min]	saline	17 ± 9	24 ± 11
	cremophor	17 ± 9	18 ± 9
	paclitaxel	23 ± 8	30 ± 12
Venular leukocyte rolling [%]	saline	21 ± 30	18 ± 15
	cremophor	43 ± 29	17 ± 13
	paclitaxel	37 ± 20	30 ± 15
Body weight [g]	saline	23 ± 2	23 ± 2
	cremophor	25 ± 3	25 ± 2
	paclitaxel	23 ± 1	24 ± 3

Fig. 3 Quantitative analysis of von Frey filament testing (0,02–2,00 g) in saline (yellow symbols), cremophor (red symbols) and paclitaxel (blue symbols) treated hairless SKH1-hr mice. Panels represent the response frequency (in %) on day 15 (left panel), 17 (middle panel) and 21 (right panel) in all animals studied. Values are given as mean ± SD. Two-way ANOVA following Tukey’s multiple comparisons test revealed the following statistically significant differences of the paclitaxel group with: **** p < 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 vs. saline; ^#### p < 0.0001, ^## p ≤ 0.01 vs. cremophor.

Intravital fluorescence microscopic analysis

Results of intravital microscopy and microscopic cell analyses are presented in [Table 1] and depicted in [Fig. 4]. There was a significant decrease in functional capillary density already after the first administration of paclitaxel on day 0 compared to the cremophor and saline treated animals ([Fig. 4] a). On day 11, a further slight decrease of functional capillary density in paclitaxel-treated animals was found ([Fig. 4] a). As sign of an inflammatory response to paclitaxel, venular leukocyte adherence was significantly increased in the paclitaxel group on day 0 and on day 11 as opposed to the cremophor and the saline group ([Fig. 4] b). In this context, venular leukocyte flux and venular leukocyte rolling, presented in [Table 1], showed no significant differences. As further sign of inflammation intravital microscopy revealed increased macromolecular leakage exclusively in the paclitaxel group indicating elevated venular endothelial permeability ([Fig. 4] c). Quantification of cells exhibiting condensation or fragmentation of nuclear chromatin resulted in a significantly higher number of apoptotic cells in the paclitaxel compared to the cremophor and the saline group ([Fig. 4] d). Again, this effect was further enhanced on day 11 in the treatment group. Quantitative analysis of other microcirculatory parameters, such as arteriolar and venular diameter are shown in [Table 1] and revealed no significant difference along the experiment and between treatment groups.

Fig. 4 Quantitative analysis of intravital fluorescence microscopy with a functional capillary density (in cm/cm²) and b venular leukocyte adherence (n/mm²) as well as c macromolecular leakage and d bisbenzimide-stained condensed nuclei (as n/HPF) at the ear of saline (yellow bars), cremophor (red bars) and paclitaxel (blue bars) treated hairless SKH1-hr mice. IVM was performed on day 0 directly after the first application and on day 11 after the sixth application of either saline, cremophor or paclitaxel. Values are given as median with 25th and 75th percentiles and whiskers indicating minimal and maximal values. Two-way ANOVA following Tukey’s multiple comparisons test revealed the following statistically significant differences of the paclitaxel group with: **** p < 0.0001 vs. saline; ^#### p < 0.0001, ^### p < 0.001, ^## p ≤ 0.01, vs. cremophor.

Multiplex assay

All blood biomarkers were assessed at the end of the experiment on day 21 in the 31 mice that underwent all tests and treatments ([Fig. 1] a). These quantitative analyses, particularly those of the inflammatory parameters, were carried out in the second treatment group ([Fig. 1] b) with n = 30 directly after the last chemotherapy on day 11. Plasma levels of IL-6 ([Fig. 5] a), IP-10 ([Fig. 5] b), MCP-1 ([Fig. 5] c) and NF-L ([Fig. 5] d) were significantly higher in the paclitaxel group than in the saline group on day 11 and decreased towards the limit of detection at day 21. BDNF plasma levels ([Table 2]) were found significantly decreased in the paclitaxel group at day 11. On day 21 BDNF plasma levels averaged ≈ 0.5 pg/ml without differences between groups. IL-1β plasma levels were found with low concentrations < 10 pg/ml on day 11 and 21 with differences between paclitaxel group vs. cremophor group at day 11 ([Table 2]). TNF-α plasma levels were found with low concentrations < 100 pg/ml on day 11 and 21 without differences between groups ([Table 2]). The analysis of VEGF-A, MMP-9 and MIP-1α revealed no relevant any notable changes at either time point or between groups ([Table 2]). C-peptide levels were below the detection limit (data not shown).

Fig. 5 Quantitative analysis of plasma concentrations of a IL-6, b IP-10, c MCP-1 and d NF-L on day 11 and day 21 in saline (yellow bars), cremophor (red bars) and paclitaxel (blue bars) treated animals. Values are given as median with 25th and 75th percentiles and whiskers indicating minimal and maximal values. Two-way ANOVA following Tukey’s multiple comparisons test revealed the following statistically significant differences of the paclitaxel group with: **** p < 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * vs. saline; ^#### p < 0.0001, ^## p ≤ 0.01, ^# p < 0.05 vs. cremophor.

Table 2 Blood biomarker concentrations in plasma of hairless SKH1-hr mice were treated with either saline, cremophor or paclitaxel. Single- and multiplex analysis were performed on day 11 (end of treatment) and on day 21 at the end of the whole experimentation time. BDNF, brain-derived neurotrophic factor; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor α; VEGF-A, vascular endothelial growth factor A; MMP-9, matrix-metallopeptidase 9; MIP-1α=CCL3, macrophage inflammatory protein 1alpha. Values are given as mean ± SD, Data were analyzed by Kruskal–Wallis test followed by Bonferroni correction which revealed the following statistically significant differences: * p < 0.05 vs. saline; # p < 0.05 vs. cremophor.

Parameter	Groups	Day 11	Day 21
BDNF [pg/ml]	saline	2.8 ± 1.4	0.4 ± 0.3
	cremophor	3.9 ± 1.8	0.6 ± 0.5
	paclitaxel	2.1 ± 0.7*#	0.7 ± 0.5
IL-1β [pg/ml]	saline	4.4 ± 0.9	1.6 ± 0.3
	cremophor	2.6 ± 0.7	1.6 ± 0.2
	paclitaxel	4.9 ± 3.0#	1.7 ± 0.7
TNF-α [pg/ml]	saline	47.5 ± 5.8	49.7 ± 18.2
	cremophor	30.5 ± 8.5	37.7 ± 9.2
	paclitaxel	52.1 ± 12.8	34.3 ± 3.7
VEGF-A [pg/ml]	saline	19.3 ± 1.6	34.5 ± 9.2
	cremophor	19.5 ± 3.0	24.9 ± 7.0
	paclitaxel	19.1 ± 2.3	39.9 ± 17.4
MMP-9 [pg/ml]	saline	23.9 ± 4.6	0.9 ± 0.6
	cremophor	21.3 ± 3.4	1.0 ± 0.6
	paclitaxel	24.9 ± 8.1	1.6 ± 1.4
MIP-1α [pg/ml]	saline	1.5 ± 0.6	–
	cremophor	1.2 ± 0.4	–
	paclitaxel	1.5 ± 0.5	–

Histology and immunohistochemistry

Representative images and data from day 21 of the morphological tests are given in [Fig. 6]. We started our analyses by quantifying the neurodegenerative effects of paclitaxel on left hind paws axon density in cross sections of nerves from paclitaxel, saline and cremophor treated mice. Treatment with paclitaxel did not cause any relevant change in axon areas (paclitaxel 2.08 µm² ± 0.16, saline 2.13 µm² ± 0.75, cremophor group 2.40 µm² ± 0.46;) and in myelin areas (paclitaxel 2.20 µm² ± 0.24, saline 2.10 µm² ± 0.54, cremophor group 2.27 µm² ± 0.36) in the different treatment groups. However, the ratio myelin/axon area showed slightly higher values in the paclitaxel group ([Fig. 6] a). Next, we examined the morphological effects of paclitaxel on axon density by immunohistochemical staining with PGP9.5. There was a decrease of approximately 45 % in both the paclitaxel and cremophor group when compared to saline group (Fig. 6b). Further, CD11b and acetylated tubulin immunohistochemistry for visualization of granulocytes and acetylated tubulin did not reveal any differences between groups ([Fig. 6] c, d). Unfortunately, the fixation of the dissected sciatic nerve was associated with many artifacts, which did not allow adequate analysis.

Fig. 6 Representative images of plantar peripheral nerves of the left hind paw in saline, cremophor and paclitaxel treated hairless SKH1-hr mice: a toluidine and nuclear red staining: the arrows indicate in panel a morphologically altered axons, which we refer to as condensed. As well as immunohistochemical staining of b PGP9.5 and quantitative analysis of PGP9.5 stained specimen with PGP9.5 positive area c CD11b: images of immune cells stained with the antibody CD11b and d acetylated tubulin at day 21. Bars indicate 5 and 10 µm. Values are given as median with 25th and 75th percentiles and whiskers indicating minimal and maximal values. One-way ANOVA followed by Sidak’s multiple comparisons test revealed the following statistically significant differences of the paclitaxel group with: ^# < 0.05 vs. cremophor.

A total of 47 animals were started in arm A ([Fig. 1] a) and 31 of these could be completely analyzed. 16 animals died during the second anesthesia for intravital microscopy from respiratory and circulatory arrest after completion of treatment on day 11. The number of deaths was evenly distributed among the groups. Since we already observed highly significant changes in our main target variable, i.e. functional capillary density in the paclitaxel group in the interim analysis after 47 animals, it was decided not to perform any further series of experiments. In arm B of the experiment, all 30 animals included could also be evaluated without loss of animals.

Discussion

In the present study, we were able to demonstrate that administration of the chemotherapeutic agent paclitaxel leads – besides the known direct cytotoxic effect – to an acute disturbance of the peripheral microcirculation in parallel to an inflammatory response. Both features were associated with significant peripheral neurological deficits from day 17 onwards, corresponding to CIPN.

The main motivation to investigate paclitaxel was because of the broad use of the taxanes for a wide variety of tumor entities, especially for gynecological tumors and the known high incidence of CIPN in association with paclitaxel [4] [51] [52]. For our experiments, we chose 30 mg/kg as the cumulative dose, since a significant CIPN can be induced with this dose [25] [53]. The primary causative factor for CIPN was previously thought to be direct cytotoxic effects on neuronal cytoskeletal structures [54].

The principal consideration that the development of CIPN under treatment with paclitaxel may be also dependent on the microcirculation became apparent from the clinical observation of patients under chemotherapy with this agent: They often present with impressive livid changes in skin color immediately during or within the first hours after the application of this chemotherapeutic agent. Also, first clinical and experimental data from different pathophysiological conditions point toward an interaction of changes in peripheral skin microcirculation and the induction of polyneuropathies. For example, in diabetic neuropathy, a correlation between disease severity and peripheral skin blood flow was described [55] [56]. However, it must be differentiated that, in contrast to CIPN, the development of diabetic neuropathy is a long-term chronic process. Microcirculatory disturbances can also be observed when other chemotherapeutic agents are used. For example, an increase in cardiac capillary permeability could be shown in heart tissue as an expression of microvascular disruption following anthracycline treatment in animal models [57]. Imaging studies have also shown a decrease in capillary density during anthracycline treatment in humans [58]. Moreover, inflammation-related endothelial damage with endothelial dysfunction and hypercoagulability were described in the context of chemotherapy-associated cardiomyopathy [59]. However, the microvascular response after paclitaxel administration especially in the symptomatic areas of CIPN has not been completely understood. To investigate this complex pathophysiological phenomenon, we chose to perform an in vivo experiment with the SKH1-hr strain, because the fingerprint-like angioarchitecture of the auricle makes this strain a powerful and cost-effective model for microvascular research [33]. Thus, artificial vessel labeling was not necessary to capture the repeated analyses of identical microvessels over a 12-day period. Using intravital microscopy on the ear of the mice one hour after application of chemotherapy on cycle days one and six, we were able to demonstrate a significant decrease in functional capillary density of paclitaxel-treated animals compared to the control and vehicle groups. We assume that this microvascular effect demonstrated in the ear of the mice also occurs under chemotherapy in the area of the paws. Two mechanisms may be responsible for this effective reduction in perfusion: obstruction or vasoconstriction. The effects of cytostatic drugs on blood flow have also been described with other oncological therapies: In patients under oxaliplatin therapy, the formation of neutrophil extracellular traps (NETs) was held responsible for the decreased blood flow with microcirculatory disturbance and an increased tendency to thrombosis. In this context, Wang et al. defined CIPN as a primary ischemic disease [60]. The other possible pathomechanism of paclitaxel-mediated endothelial toxicity was described via its direct effect on vasomotor tone mediated by the microscopic vascular nerve reticulum [61] [62]. Vassilakopoulou et al. were also able to demonstrate a reduction in endothelium-dependent flow-mediated dilation (FMD) in the brachial artery in patients treated weekly with paclitaxel. The decrease in FMD causes a reduced ability of the blood vessels to dilate when blood flow increases and the endothelium is active. In this study, however, the reduction in FMD was not associated with an increase in inflammatory parameters such as plasma levels of IL-6 and TNF-α [63]. In the process of peripheral nerve injury, the pathophysiologic processes of reperfusion syndrome following vasoconstriction or obstruction may also be critical to the aggravation of CIPN. Reperfusion causes calcium overload, the formation of reactive oxygen species (ROS) and the subsequent activation of the mitochondrial permeability transition pore (MPT), which then contributes significantly to reperfusion injury [64]. In line with this, we observed both endothelial leakage as an expression of increased vascular permeability in the paclitaxel group and systemic inflammation reflected by a significant increase of the proinflammatory cytokine IL-6. This combination points toward direct toxicity of paclitaxel at the endothelium of the vessels leading to an inflammatory reaction. Leukocyte adherence at the venular endothelium was described as an expression of the direct toxic effects and inflammation induction on endothelial cells [37]. Along this, we observed by intravital microscopy a significantly increased venular leukocyte adherence in the paclitaxel group by intravital microscopy. The significantly increased plasma levels of IL-6 and IP-10, exclusively on day 11 reflect the inflammatory component with a direct toxic effect of paclitaxel. We chose to determine IP-10 because this protein is produced exclusively by interferon IFN-γ contact with macrophages, monocytes, and endothelial cells. IP-10 causes an enhancement of the expression of various adhesion molecules on endothelia and inhibits angiogenesis besides stimulating monocytes, NK cells, and T cells to migrate into the tissue. This increase in turn explains the significantly increased venular leukocyte adherence from the point of view of the local toxicity of paclitaxel on the endothelium. We observed a large dispersion of IP-10 in the cremophor group together with slightly increased venular leukocyte adherence as evidence for inflammation induction by cremophor. The significant increase of MCP-1 (synonymous with CCL2) in the paclitaxel group at day 11 may indicate the recruitment of immune cells especially monocytes as a sign of neuroinflammation [41]. This is coherent with the observation of Thacker et al., who identified CCL2 deficient mice being resistant to the induction of sensory neuropathies [65]. In addition to this pathomechanism, MCP-1 was also associated with oxidative stress in several studies. For example, oxidative stress with escalated MCP-1 expression in rats was described in an ischemia and reperfusion model [66].

We found no relevant increase of VEGF-A, MMP9 and MIP-1α in any of our animals. However, we found significantly lower plasma levels of BDNF in paclitaxel treated mice. This is consistent with the work of Azoulay et al. who demonstrated that this neuronal growth factor may play a critical predisposing role in the survival and repair of injured neurons [67]. The significant increase in plasma levels of NF-L in paclitaxel-treated animals on day 11 emphasizes the clinical severity of CIPN. Increased plasma levels of NF-L in vitro were already described for preclinical studies in rats [68] [69] and also in patients with breast or ovarian cancer treated with paclitaxel [70].

Further, multiple mechanisms of direct neurotoxicity and inflammation, particularly of axonal damage, were described as primary cause for CIPN [71]. This comprised alterations of axonal transport [17], mitochondrial function [72] [73] and ion channel function with consequences on Ca²⁺ homeostasis [74], neuroinflammation [75], and direct DNA damage. This is in line with our intravital microscopic findings with clear signs for an activation of apoptosis already early after administration of the first cycle of paclitaxel. The strengths of our study lie in its comprehensive investigation of microcirculatory disturbances following paclitaxel application and their impact on the organism, assessed through clinical neurophysiology, symptomatology, serological markers, and cellular changes. However, some limitations have to be mentioned. One limitation of this study is the fact that no animal model can fully represent the entire clinical spectrum due to evolutionary differences between species. In our study, we focused on animal models of CIPN and compared them based on outcome measures related to allodynia, hyperalgesia, and neurophysiological changes in nerve function. However, many patients also report additional symptoms, such as tingling and persistent pain, which are not fully captured in these models. Ideally, animal models would mimic all human symptoms, but such models do not exist. In contrast to the clinical setting, we decided to apply paclitaxel via the intraperitoneal route rather than intravenously. The intraperitoneal route is commonly used in small animal models [25]. A further limitation is the use of the SKH-1 mouse strain. This strain has been used to study the peripheral microcirculation in the ear under paclitaxel administration. No data exist on CIPN models in the paw region in this mouse strain.

Although these mechanisms seem to play a prominent role for the development of CIPN, it is an intriguing question why there is such a high variability in patients developing CIPN with long-term consequences or not. Our results strengthen the evidence that besides direct cytotoxic effects of paclitaxel, also reperfusion-induced inflammation and direct microcirculatory disturbances play an important role in CIPN. The proposed prevention strategy of limb cooling and/or compression has so far been explained solely by reducing the load of chemotherapy drugs, and thus their direct cytotoxic effects to the periphery [76] [77]. However, it is not clear yet, if, and to what extent temperature changes directly modulate the inflammatory response due to reperfusion, which we described here. The same accounts for the effect of compression of the perineural microcirculation. Thus, to improve this preventive approach further systematic experimental and clinical investigation is needed.

Conclusion

Our results suggest a close interplay between the development of CIPN with microcirculatory dysfunction, accompanied by inflammation during treatment with paclitaxel.

This knowledge could also form the basis for prophylactic or at least soothing supportive treatment strategies for CIPN.

Data Availability Statement

Data are available from the authors upon reasonable request.

Conflict of Interest

Susanne Reuter: Advisory board MSD 2023. The other authors have declared that there are no conflicts of interest in association with this study.

Acknowledgement

Many thanks to Christine Schlie for excellent support during the series of experiments and to Dorothea Frenz for the histological processing of the tissue samples. The work was supported by the Medical Research Council of University Medical Center Rostock (grant no. 889043). The authors thank them for this support. The founders had no role in study design, data collection or analysis.

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Publication History

Received: 29 August 2024

Accepted after revision: 30 November 2024

Article published online:
17 March 2025