Imaging Arm Regeneration: Label-Free Multiphoton Microscopy to Dissect the Process in Octopus vulgaris

Abstract

Cephalopod mollusks are endowed with an impressive range of features that have captured the attention of scientists from different fields, the imaginations of artists, and the interests of the public. The ability to spontaneously regrow lost or damaged structures quickly and functionally is among one of the most notable peculiarities that cephalopods possess. Microscopical imaging techniques represent useful tools for investigating the regenerative processes in several species, from invertebrates to mammals. However, these techniques have had limited use in cephalopods mainly due to the paucity of specific and commercially available markers. In addition, the commonly used immunohistochemical staining methods provide data that are specific to the antigens studied. New microscopical methods were recently applied to vertebrates to investigate regenerative events. Among them, multiphoton microscopy appears promising. For instance, it does not depend on species-related epitopes, taking advantage of the specific characteristics of tissues and allowing for its use in a species-independent way. Here, we illustrate the results obtained by applying this label-free imaging technique to the injured arm of Octopus vulgaris, a complex structure often subject to injury in the wild. This approach allowed for the characterization of the entire tissue arm architecture (muscular layers, nerve component, connective tissues, etc.) and elements usually hardly detectable (such as vessels, hemocytes, and chromatophores). More importantly, it also provided morpho-chemical information which helped decipher the regenerative phases after damage, from healing to complete arm regrowth, thereby appearing promising for regenerative studies in cephalopods and other non-model species.

Deciphering regeneration through non-model animals: A century of experiments on cephalopod mollusks and an outlook at the future

“…Recently, cephalopod arm wound healing was subjected to closer investigation (Shaw et al, 2016): wound closure was followed for the first 24 h after amputation in O. vulgaris, using classical histological staining, immunohistochemistry (IHC), high-resolution ultrasound imaging and electron microscopy. Despite the diverse experimental settings (water temperature, animals' age, species, sex, surgical method, site of lesion) the newer studies (Tressler et al, 2014;Shaw et al, 2016;Imperadore et al, 2022) confirm the earlier macro-and microscopic accounts, and especially the key role of amoebocytes/hemocytes (Polglase et al, 1983;Imperadore, et al, 2022). Shaw et al (2016) also suggested i. a role for muscles cells in plug development, ii.…”

“…The different settings, however, may influence events, pathways, and mechanisms underlying healing and regeneration. How the problem of ecological validity can be profitably overcome, is suggested by a recent work on O. vulgaris ( Imperadore et al, 2022 ). The study took advantage of the high incidence of damaged wild-caught animals for this species, reported to occur at a rate of around 51% in the Gulf of Naples ( Florini et al, 2011 ) and apparently linked to sublethal predation, autophagy, mating and aggressive behaviours (see Imperadore and Fiorito, 2018 ) for testing label-free multiphoton microscopy in the investigation of regeneration in cephalopods (see below).…”

“…Imaging regeneration has also proved advantageous, in several species, to investigate regeneration. Despite limited access to commercial markers or techniques for real time imaging, some tools have recently been developed: label-free multiphoton microscopy ( Imperadore et al, 2018 ; Imperadore et al, 2022 ), 18F-FDG PET ( Zullo et al, 2018 ), optimized CUBIC clearing protocol ( Deryckere et al, 2020 ) and neural tracing ( Imperadore et al, 2019a ; Imperadore et al, 2019b ).…”

Vibrational spectroscopy and multiphoton microscopy for label-free visualization of nervous system degeneration and regeneration

“…In experimental neuroscience, the omission of markers allows for better comparison of preclinical data across different animal species, as the obtained information is independent of staining protocols specific to each model. For example, multimodal label-free multiphoton microscopy combining CARS, TPEF, and SHG imaging has been used in octopus PNS injury to examine the mechanisms of degeneration processes, scarring and subsequent regeneration of the pallial nerve and of the whole arm (Imperadore et al 2018 , 2022 ).…”

The salamander blastema within the broader context of metazoan regeneration

“…Beyond the salamander limb field, the term “blastema” has been applied more generally to describe a large variety of structures, in many species, during the regeneration of several different organ systems ( Reddien and Sánchez Alvarado, 2004 ; Bely and Nyberg, 2010 ; Fernando et al, 2011 ; Tanaka and Reddien, 2011 ; Bradshaw et al, 2015 ; Sallin et al, 2015 ; Zhao et al, 2016 ; Imperadore et al, 2017 ; 2022 ; Elchaninov et al, 2021 ; Bando et al, 2022 ; Vonk et al, 2022 ). These structures are generally superficially similar in that they consist of a mass of cells which must proliferate and repattern itself to regenerate large anatomical structures ( Bely and Nyberg, 2010 ; Tanaka and Reddien, 2011 ; Zhao et al, 2016 ; Elchaninov et al, 2021 ) .…”