Biomedical subjects

G Terenghi

Publications and source records attributed to G Terenghi.

At least 19 recordsLinked to original sources

PubMed2006

End-to-side nerve coaptation: a qualitative and quantitative assessment in the primate.

There are several reasons why end-to-side nerve coaptation has not been widely adopted clinically. Among these are the putative damage inflicted on the donor nerve and the variable quality of the regeneration in the recipient nerve. So far experiments on end-to-side nerve repair have been short term and mostly carried out on rats. This long-term study of end-to-side nerve repair of ulnar to median and median to ulnar nerve was performed using adult nonhuman primates. Eleven nerve repairs were studied at different time points. Eighteen, 22, 33 and 57 months after surgery a qualitative and quantitative analysis of the donor nerve and regenerating nerve revealed variable levels of percentage axonal regeneration compared with matched controls (1.4%-136%). Morphological evidence of donor nerve damage was identified distal to the coaptation site in four of the 11 cases, and in these cases the best axonal regeneration in the corresponding recipient nerves was observed. This donor nerve damage could neither be demonstrated in terms of a decrease in axon counts distal to the coaptation nor as donor target organ denervation. Recipient target organ regeneration like the axonal regeneration varied, with evidence of motor regeneration in eight out of 11 cases and sensory regeneration, as measured by percentage innervation density compared with matched controls, varied from 12.5% to 49%. Results from the present study demonstrate that the end-to-side coaptation technique in the nonhuman primate does not give predictable results. In general the motor recovery appeared better than the sensory and in those cases where donor nerve damage was observed there was better motor and sensory regeneration overall than in the remaining cases.

Animals↗

PubMed2006

The reinnervation pattern of wounds and scars may explain their sensory symptoms.

Anaesthesia, pruritus and pain are common in cutaneous scars. The reinnervation pattern of healing wounds and scars might help to explain these symptoms, as sensory neurotransmitters are known to be mediators of inflammation and healing. We quantified the regeneration patterns of blood vessels and nerves in excisional skin wounds as they matured into scars. Mice underwent 1cm(2) full thickness skin excisions. Wounds were harvested between five and 84 days. Sections underwent immunohistochemical staining for protein gene product 9.5 (PGP9.5) a pan-neuronal marker, and the sensory neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP). The endothelial marker von Willebrand factor (VWF) was used to allow co-localisation and quantification of blood vessels. Nerve fibre density was quantified at multiple sites within wounds. There was no difference in the reinnervation/revascularisation pattern between peripheral and central sites. The density of PGP9.5, CGRP, SP and VWF peaked between 14 and 42 days, and levels of PGP9.5, CGRP and VWF all decreased to approximately those found in unwounded skin by 84 days (mature scar). SP levels, however, remained elevated at approximately twice the density found in unwounded skin. Increased densities of SP and CGRP in healing wounds could explain the unpleasant sensory symptoms of healing wounds.

Animals↗

PubMed2006

Neuronal survival, regeneration and musclemorphology after posterior C7 nerve transfer: an experimental study.

C7 nerve transfer has been widely used in treating brachial plexus avulsion injuries. Little is known regarding the survival and regeneration of C7 motor and sensory neurons including their morphological changes after this procedure and also the possible change of muscle fibre phenotype. In this experimental study, the posterior division of C7 nerve was transferred to the musculocutaneous nerve ipsilaterally, and using fluorescent tracing techniques, the C7 spinal cord segment and dorsal root ganglion were found to contain 630.9 +/- 86.7 motor neurons and 3916.0 +/- 517.3 sensory neurons, respectively. Six months following transfer, 90% of the motor neurons and 78% of the sensory neurons survived and approximately 40% of them had regenerated and all displayed normal soma size. After posterior C7 transfer and reinnervation, the target muscles showed a percentage pattern of distribution and mean fibre diameters similar to those seen in normal biceps muscle. The present study suggests that the posterior C7 nerve transfer provides sufficient number of neurons and satisfactory results for regeneration to obtain an acceptable functional recovery.

Animals↗

PubMed2006

The role of neuropeptides in psoriasis.

The pathogenesis of psoriasis is incompletely understood but cutaneous neurogenic inflammation is probably involved. This involvement is suggested by a number of clinical and histological observations. Reports about the distribution of cutaneous nerves and the quantification of nerve growth factor and neuropeptides, including calcitonin gene-related peptide and vasoactive intestinal peptide, in lesional and nonlesional psoriatic skin suggest that sensory neuropeptides contribute to the development of psoriasis. This review summarizes what is known about the role of neurogenic markers in psoriasis.

Calcitonin Gene-Related Peptide↗

PubMed2005

Nerve fibre and sensory end organ density in the epidermis and papillary dermis of the human hand.

Quantification of sensory recovery after peripheral nerve surgery is difficult and no accurate techniques are available at present. Quantification of reinnervated skin has been used experimentally, and in some clinical studies, but the lack of knowledge about the normal sensory distribution has been a problem. The purpose of this study was, therefore, to map the density of sensory end organs, nerve fibres and free nerve endings in the glabrous skin of the human hand. Skin biopsies were taken from patients undergoing acute and elective hand surgery. Nerve fibres were stained in the epidermis and papillary dermis and quantified in five sites on the palm of the hand, using protein gene product 9.5 immunoreactivity-a panneuronal marker. The finger tip skin was found to have more than twice the nerve fibre density in the papillary dermis than the skin of the palm, and the number of Meissner corpuscles in the finger tip was also higher than in the palm. We found a reduction in innervation density with increasing age in the dermis, however, that was not the case for the epidermis. The innervation of the epidermis showed high interindividual variability and unlike the papillary dermis did not display any pattern of distribution in the hand.

Adult↗

PubMed2005

Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve.

Adhesion molecules are important in supporting axonal regeneration. Qualitative studies have described increased expression of neural cell adhesion molecule (NCAM) and N-cadherin in models of nerve injury allowing active regeneration. In this study we have used quantitative immunohistochemistry to compare expression of NCAM and N-cadherin after nerve injury either with active regeneration (crush) into the distal stump or without (axotomy and capping). Quantification was performed 15 days after axotomy in proximal and distal stumps. Quantification after crush either proximal, distal or within the crushed area was performed at 2, 7, 15 and 30 days after injury. Axotomy induced increases in expression in proximal stumps between two and three times those in uninjured nerves for both molecules. In distal stumps, N-cadherin levels increased seven-fold, yet NCAM levels did not exceed control values. After crush, NCAM immunoreactivity increased in the crushed area and distal stump in contrast to axotomy and NCAM-positive axons co-localized with PGP9.5. N-cadherin levels in the distal stump increased above control levels, but the magnitude of the increase seen after crush was different to those seen after axotomy. In conclusion, increased expression of adhesion molecules, particularly NCAM, in the distal stump of injured nerves is dependent upon the presence of regenerating axons.

Animals↗

PubMed2004

Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.

Acetylcysteine↗

PubMed2003

Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit.

The clinical outcome of peripheral nerve injuries remains disappointing, even in the ideal situation of a primary repair performed with optimal microsurgical techniques. Primary repair is appropriate for only about 85% of injuries, and outcome is worse following secondary nerve repair, partly owing to the reduced regenerative potential of chronically axotomised neurons. Leukaemia inhibitory factor (LIF) is a gp-130 neurocytokine that is thought to act as an 'injury factor', triggering the early-injury phenotype within neurons and potentially boosting their regenerative potential after secondary nerve repair. At 2-4 months after sciatic nerve axotomy in the rat, 1 cm gaps were repaired using either nerve isografts or poly-3-hydroxybutyrate conduits containing a calcium alginate and fibronectin hydrogel. Regeneration was determined by quantitative immunohistochemistry 6 weeks after repair, and the effect of incorporating recombinant LIF (100 ng/ml) into the conduits was assessed. LIF increased the regeneration distance in repairs performed after both 2 months (69%, P=0.019) and 4 months (123%, P=0.021), and was statistically comparable to nerve graft. The total area of axonal immunostaining increased by 21% (P>0.05) and 63% (P>0.05), respectively. Percentage immunostaining area was not increased in the 2 months group, but increased by 93% in the repairs performed 4 months after axotomy. Exogenous LIF, therefore, has a potential role in promoting peripheral nerve regeneration after secondary repair, and can be effectively delivered within poly-3-hydroxybutyrate bioartificial conduits used for nerve repair.

Animals↗

PubMed2003

Increased innervation of the vulval vestibule in patients with vulvodynia.

BACKGROUND: Vulval vestibulitis is a condition characterized by the sudden onset of a painful burning sensation, hyperalgesia, mechanical allodynia, and occasionally pruritus, localized to the region of the vulval vestibulus. It is considered the commonest subset of vulvodynia. Pain precipitated in the absence of nociceptor stimuli might be triggered by previous peripheral nerve injury, or by the release of neuronal mediators, which set off inappropriate impulses in nonmyelinated pain fibres sensitizing the dorsal horn neurones. The pathophysiology of vulval vestibulitis is still unclear. OBJECTIVES: The objective of this study was to evaluate the nerve fibre density and pattern, in specimens of vulval vestibulus, in normal subjects and in patients with vestibulitis, and provide objective diagnostic criteria for this condition. Methods Twelve patients with a history of the vestibulitis type of vulvodynia, and eight normal subjects underwent biopsy of the posterior wall of the vulval vestibule. Quantitative immunohistochemistry was performed, using antisera to the general neuronal marker protein gene product (PGP) 9.5, and to the neuropeptide calcitonin gene-related peptide (CGRP), on 15- microm sections. RESULTS: There was a statistically significant increase of density and number of PGP 9.5 immunoreactive in the papillary dermis of patients with vulvodynia of the vestibulitis type, compared with those of controls. However, the distribution pattern of the innervation showed no significant change. There were no significant differences in CGRP staining between patients and controls. CONCLUSIONS: It is concluded that the increase of PGP 9.5 immunoreactive nerve fibres, in patients with vulvodynia, may be either secondary to nerve sprouting, or may represent neural hyperplasia. Increased innervation may be applied as an objective diagnostic finding in vulval vestibulitis syndrome.

Adult↗

PubMed2003

A composite poly-hydroxybutyrate-glial growth factor conduit for long nerve gap repairs.

There is considerable evidence that peripheral nerves have the potential to regenerate in an appropriate microenvironment. We have developed a novel artificial nerve guide composed of poly 3-hydroxybutyrate (PHB) filled with glial growth factor (GGF) suspended in alginate hydrogel. Gaps of 2-4 cm in rabbit common peroneal nerve were bridged using a PHB conduit containing either GGF in alginate hydrogel (GGF) or alginate alone (Alginate), or with an empty PHB conduit (Empty). Tissues were harvested 21, 42 and 63 days post-operatively. Schwann cell and axonal regeneration were assessed using quantitative immunohistochemistry. At 21 days, addition of GGF increased significantly the distance of axonal and Schwann cells regeneration in comparison with that observed in Alginate and Empty conduits for both gap lengths. The axons bridged the 2-cm GGF conduits gap by 63 days, with a comparable rate of regeneration seen in 4-cm conduits. Schwann cells and axonal regeneration quantity was similar for both gap lengths in each group. However, at all time points the quantity of axonal and Schwann cells regeneration in GGF grafts was significantly greater than in both Alginate and Empty conduits, the latter showing better regeneration than Alginate conduits. The results indicate an inhibitory effect of alginate on regeneration, which is partially reversed by the addition of GGF to the conduits. In conclusion, GGF stimulates a progressive and sustainable regeneration increase in long nerve gap conduits.

Alginates↗

PubMed2002

Poly-3-hydroxybutyrate (PHB): a resorbable conduit for long-gap repair in peripheral nerves.

The repair of peripheral nerve injuries with an autologous nerve graft is still the treatment of choice, although it is widely recognised that the method is associated with donor-site morbidity and a suboptimal functional outcome. An alternative approach is to use a nerve conduit to provide a guidance channel for the regenerating nerve. This study investigates the potential of poly-3-hydroxybutyrate (PHB) nerve conduits to bridge long nerve gaps (up to 4 cm) in a rabbit common-peroneal-nerve injury model. Regeneration was assessed up to 63 days postoperatively, and compared with that achieved using nerve autografts. By 42 days, regenerating axons had bridged nerve gaps of all lengths in groups with nerve autografts and in those with PHB conduits. The regeneration distance increased with time but did not vary with gap length (P </= 0.009, 14 versus 21 days, PHB tube 2 cm, 3 cm and 4 cm, Mann-Whitney U -test). At 42 days, the area of immunostained regenerating fibres in the PHB group was greater than that in the nerve autograft group (P < 0.001, PHB versus autograft, 21 and 42 days, three-way analysis of variance (ANOVA)). These results suggest that PHB conduits support peripheral nerve regeneration up to 63 days, and that they are suitable for long-gap nerve injury repair.

Analysis of Variance↗

PubMed2001

Retroviral labeling of Schwann cells: in vitro characterization and in vivo transplantation to improve peripheral nerve regeneration.

Transplantation of Schwann cells (SCs) is a promising treatment modality to improve neuronal regeneration. Identification of the transplanted cells is an important step when studying the development of this method. Genetic labeling is the most stable and reliable method of cell identification, but it is still unclear whether it has deleterious effect on SC characteristics. Our aim was to achieve a stable population of SCs transduced with the lacZ gene at a high frequency using a retroviral vector in vitro, and to follow the labeled SC in vitro to assess their viability and phenotypic marker expression. Furthermore, we transplanted lacZ-labeled SCs in a conduit to repair peripheral nerve to investigate their effect on nerve regeneration in vivo. Rat and human SCs were cultured and transduced with an MFG lacZ nls marker gene, achieving a transduction rate of 80% and 70%, respectively. Rat SCs were kept in culture for 27 weeks and examined every 4 weeks for expression of lacZ, viability, and phenotypic marker expression of GFAP, p75, MHC I and II. Throughout this period, transduced rat SCs remained viable and continued to proliferate. The proportion of cells expressing lacZ dropped only by 10% and the expression of phenotypic markers remained stable. Transduced human SCs were followed up for 4 weeks in culture. They proliferated and continued to express the lacZ gene and phenotypic marker expression of GFAP and p75 was preserved. Primary culture of transduced rat SCs were transplanted, syngeneically, in a conduit to bridge a 10 mm gap in sciatic nerve and the grafts were examined after 3 weeks for the presence and participation of labeled SCs and for axonal regeneration distance. Transplanted transduced rat SCs were clearly identified, taking part in the regeneration process and enhancing the axonal regeneration rate by 100% (at the optimal concentration) compared to conduits without SCs. Thus, retroviral introduction of lacZ gene has no deleterious effect on SCs in vitro and these SCs take part and enhance nerve regeneration in vivo.

Animals↗

PubMed2001

Immunolocalisation of sodium channel NaG in the intact and injured human peripheral nervous system.

The voltage-gated 'glial' sodium channel NaG belongs to a distinct molecular class within the multi-gene family of mammalian sodium channels. Originally found in central and peripheral glia, NaG has since been detected in neurons in rat dorsal root ganglia (DRG) and may play a role in Schwann cell-axon interactions. We have studied the presence of NaG-like immunoreactivity in the intact and injured human peripheral nervous system using a specific affinity-purified antibody. Nerve fibres in normal and injured peripheral nerves and normal skin exhibited intense NaG-immunoreactivity. Numerous NaG-immunoreactive nerve fibres surrounded neuronal cell bodies within postmortem control DRG, and in DRG avulsed from the spinal cord (i.e. after traumatic central axotomy). There were no significant differences in the pattern of NaG immunostaining between control and avulsed DRG, or with delay after injury. Generally, the neuronal cell bodies were only very weakly immunoreactive to NaG, indicating that the NaG immunoreactivity was predominantly in Schwann cells/myelin. In accord, we demonstrated NaG immunostaining in cultured human and rat Schwann cells, and in distal nerve after wallerian degeneration. NaG thus appears to be a useful new marker for Schwann cells in the human PNS, and a role in neuropathy deserves investigation.

Animals↗

PubMed2001

Increased sodium channel SNS/PN3 immunoreactivity in a causalgic finger.

The sodium channels SNS/PN3 and NaN/SNS2 are regulated by the neurotrophic factors-nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF), and may play an important role in the development of pain after nerve injury or inflammation. These key molecules have been studied in an amputated causalgic finger and control tissues by immunohistochemistry. There was a marked increase in the number and intensity of SNS/PN3-immunoreactive nerve terminals in the affected finger, while GDNF-immunoreactivity was not observed, in contrast to controls. No differences were observed for NGF, trk A, NT-3 or NaN/SNS2-immunoreactivity. While further studies are required, these findings suggest that accumulation of SNS/PN3 and/or loss of GDNF may contribute to pain in causalgia, and that selective blockers of SNS/PN3 and/or rhGDNF may provide effective novel treatments.

Aged↗

PubMed2001

A novel use of alginate hydrogel as Schwann cell matrix.

The use of bioresorbable conduits supplemented with Schwann cells (SCs) is a promising tissue engineering technique to replace nerve grafting. Alginate hydrogel (AH), as a SC tissue engineering matrix, has many advantages over previously used matrices but has not been evaluated for this purpose. In this study, the viability and proliferation of SCs together with SC function in AH was evaluated in vitro. AlamarBlue cell assay was used to monitor the viability of SCs in AH and compared to SC viability in collagen gel, fibrin glue, hyaluronic acid, Matrigel, and standard culture plate over 5 days in culture. The results showed that the viability and growth of SCs in different matrices over the culture period did not significantly differ to culture plate culture. SC function when suspended in AH was monitored using chick embryo dorsal root ganglia (CDRG) growth assay. Growth of CDRG in AH with or without SCs was compared to CDRG growth without AH matrix. After 3 days in culture, the mean length of neurite sprouting was measured. The results showed that there was neurite growth in AH but was reduced to 43% of control. The neurite growth in AH was, however, enhanced by 170% when SCs were suspended in the gel. In conclusion, AH supported SC viability and function in vitro and may be useful in peripheral nerve tissue engineering in reconstructive procedures.

Alginates↗

PubMed2000

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.

Adult↗

PubMed2000

Long-term effect of vital labelling on mixed Schwann cell cultures.

Schwann cell transplantation following neuronal injury could encourage regeneration of spinal cord as well as improving peripheral nerve gap repair. In order to gain a better understanding of the role of transplanted Schwann cells in vivo, it is essential to be able to follow their behaviour after transplantation. Our aim was to evaluate the suitability of two vital fluorescent labels on the proliferation rate and phenotypic stability of Schwann cells, in either pure culture or mixed co-culture. Primary cultures of Schwann cells were obtained from Dark Agouti and Lewis neonatal rats and labelled with H33342 and PKH26, respectively. In mixed cultures, a 50: 50 mixture of Dark Agouti and Lewis Schwann cells was present. Labelled cultured cells were examined at 1, 2 and 4 weeks for viability and phenotypic marker expression of S100, GFAP, p75, MHC I, MHC II and compared with corresponding unlabelled cells. The results showed that although there was no deleterious interaction in the mixed cultures, the viability was reduced by the labelling after 2 weeks. Labelled cells could be distinguished up to 4 weeks, but there was leakage of H33342 label after 2 weeks. Labelled Schwann cells showed reduced expression of phenotypic markers, especially p75 when labelled with H33342. In conclusion, H33342 and PKH26 can be used as fluorescent markers of Schwann cells for short-term studies, for a maximum of 2 weeks, but different markers may be needed for longer experiments.

Animals↗

PubMed2000

Differential effects of NT-3 on reinnervation of the fast extensor digitorum longus (EDL) and the slow soleus muscle of rat.

Previous studies of gastrocnemius muscle reinnervation showed specific normalization of the proportion and diameter of fast type 2b muscle fibres following NT-3 delivery to the proximal stump of the cut sciatic nerve. Here, we investigate if normalization was related to greater improvement of muscle reinnervation of fast (extensor digitorum longus; EDL) than slow (soleus) motor units. NT-3-impregnated (NT-3 group) or plain fibronectin (FN group) mats were inserted into a sciatic nerve gap. Neuromuscular junctions (NMJs) labelled with TRITC-alpha-bungarotoxin were colabelled with calcitonin gene-related peptide (CGRP) or 4E2 antisera and imaged using confocal microscopy. CGRP and 4E2 were used as markers for newly reinnervated and structurally mature NMJs, respectively. At 40 days postsurgery, denervated NMJs in EDL and soleus muscles of both groups presented a 50% decrease of surface area due to decreased width. At day 80 in EDL, more NMJs were reinnervated by CGRP-immunoreactive terminals in the NT-3 (7.1%) than in the FN group (4.2%); there was no difference between groups for soleus. At 120 days, 4E2-immunoreactive NMJs were more numerous in EDL of the NT-3 (40.0%) than in the FN group (7.3%), unlike in soleus (NT-3, 1. 6%; FN, 1.8%), and presented a partial size recovery. These results indicate that NT-3 preferentially improves reinnervation of fast muscles over slow muscle, although the mechanism of this improvement is still unclear.

Animals↗