Biomedical subjects

A M Ritter

Publications and source records attributed to A M Ritter.

At least 19 recordsLinked to original sources

PubMed2001

Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the "hairy" skin of newborn mice: early maturation of hair follicle afferents.

Adult skin sensory neurons exhibit characteristic projection patterns in the dorsal horn of the spinal gray matter that are tightly correlated with modality. However, little is known about how these patterns come about during the ontogeny of the distinct subclasses of skin sensory neurons. To this end, we have developed an intact ex vivo somatosensory system preparation in neonatal mice, allowing single, physiologically identified cutaneous afferents to be iontophoretically injected with Neurobiotin for subsequent histological analyses. The present report, centered on rapidly adapting mechanoreceptors, represents the first study of the central projections of identified skin sensory neurons in neonatal animals. Cutaneous afferents exhibiting rapidly adapting responses to sustained natural stimuli were encountered as early as recordings were made. Well-stained representatives of coarse (tylotrich and guard) and fine-diameter (down) hair follicle afferents, along with a putative Pacinian corpuscle afferent, were recovered from 2-7-day-old neonates. All were characterized by narrow, uninflected somal action potentials and generally low mechanical thresholds, and many could be activated via deflection of recently erupted hairs. The central collaterals of hair follicle afferents formed recurrent, flame-shaped arbors that were essentially miniaturized replicas of their adult counterparts, with identical laminar terminations. The terminal arbors of down hair afferents, previously undescribed in rodents, were distinct and consistently occupied a more superficial position than tylotrich and guard hair afferents. Nevertheless, the former extended no higher than the middle of the incipient substantia gelatinosa, leaving a clear gap more dorsally. In all major respects, therefore, hair follicle afferents display the same laminar specificity in neonates as they do in adults. The widely held misperception that their collaterals extend exuberant projections into pain-specific regions of the dorsal horn during early postnatal life is shown to have multiple, deep-rooted underpinnings.

Animals↗

PubMed2001

Excess target-derived neurotrophin-3 alters the segmental innervation of the skin.

It is thought that dermatomes are established during development as a result of competition between afferents of neighbouring segments. Mice that overexpress neurotrophins in the skin provide an interesting model to test this hypothesis, as they possess increased numbers of sensory neurons, and display hyperinnervation of the skin. When dermatomal boundaries were mapped in adult mice, it was found that those in nerve growth factor and brain-derived neurotrophic factor overexpressers were indistinguishable from wild-type animals but that overlap between adjacent segments was greatly reduced in neurotrophin-3 (NT-3) overexpressers. However, dermatomes in heterozygous NT-3 knockout mice displayed no more overlap than wild-types. In order to quantify differences across strains, innervation territories of thoracic dorsal cutaneous nerves were mapped and measured in adult mice. Overlap between adjacent dorsal cutaneous nerves was normal in nerve growth factor overexpressing mice, but much reduced in NT-3 overexpressers. However, this restriction was not reflected in the central projection of the dorsal cutaneous nerve, creating a mismatch between peripheral and central projections. Dorsal cutaneous nerve territories were also mapped in neonatal mice aged postnatal day 7-8. In neonates, nerve territories of NT-3 overexpressers overlapped less than wild-types, but in neonates of both strains the amount of overlap was much greater than in the adult. These results indicate that substantial separation of dermatomes occurs postnatally, and that excess NT-3 enhances this process, resulting in more restricted dermatomes. It may exert its effects either by enhancing competition, or by direct effects on the stability and formation of sensory endings in the skin.

Aging↗

PubMed2000

On the problem of lamination in the superficial dorsal horn of mammals: a reappraisal of the substantia gelatinosa in postnatal life.

Although it is one of the most distinctive and earliest recognized features in the spinal cord, the substantia gelatinosa (SG) remains among the most enigmatic of central nervous system regions. The present neuroanatomical studies employed transganglionic transport of horseradish peroxidase conjugates of choleragenoid (B-HRP) and the B4 isolectin of Bandeiraea simplicifolia (IB4-HRP) on opposite sides to compare the projection patterns of myelinated and unmyelinated cutaneous primary afferents, respectively, within the superficial dorsal horn of the spinal cord in postnatal mice, from shortly after birth to adulthood. Putative unmyelinated afferents labeled with IB4-HRP gave rise to a dense sheet of terminal-like labeling restricted to the outer half of the SG. In contrast, myelinated inputs labeled with B-HRP gave rise to a similarly dense sheet of terminal-like labeling that occupied the inner half of the SG. This adult organization, with two dense sheets of terminal labeling in the superficial dorsal horn, was clearly evident shortly after birth using these markers, prior to the emergence of the SG. Furthermore, the location of the SG proper varied considerably within the dorsoventral plane of the dorsal horn according to mediolateral and segmental locations, a finding that was also seen in comparative studies in rat and cat. These findings caution against equating the SG in particular, and the superficial dorsal horn in general, with nociceptive processing; at minimum, the SG subserves a clear duality of function, with only a thin portion of its outermost aspect devoted to pain.

Aging↗

PubMed2000

Maturation of cutaneous sensory neurons from normal and NGF-overexpressing mice.

In the rodent, cutaneous sensory neurons mature over the first two postnatal weeks, both in terms of their electrical properties and their responses to mechanical stimulation of the skin. To examine the coincidence of these events, intracellular recordings were made from neurons in the dorsal root ganglion (DRG) in an in vitro spinal cord, DRG, and skin preparation from mice between the ages of postnatal day 0 and 5 (P0-P5). We also examined mice in which nerve growth factor (NGF) is overexpressed in the skin. NGF has been shown to be involved in a number of aspects of sensory neuron development and function. Therefore we ask here whether excess target-derived NGF will alter the normal course of development, either of somal membrane properties, physiological response properties, or neuropeptide content. In wild-type mice, somal action potentials (APs) were heterogeneous, with some having simple, uninflected falling phases and some displaying an inflection or break on the falling limb. The proportion of neurons lacking an inflection increased with increasing age, as did mean conduction velocity. A variety of rapidly and slowly adapting responses could be obtained by gently probing the skin; however, due to relatively low thresholds and firing frequencies, as well as lack of mature peripheral receptors such as hairs, it was not possible to place afferents into the same categories as in the adult. No correlation was seen between the presence or absence of an inflection on the somal AP (a marker for high-threshold mechanoreceptors in adult animals) and either peripheral threshold or calcitonin-gene related peptide (CGRP) content. Small differences in the duration and amplitude of the somal AP were seen in the NGF-overexpressing mice that disappeared by P3-P5. Excess target-derived NGF did not alter physiological response properties or the types of neurons containing CGRP. The changes that did occur, including a loss of the normal relationship between AP duration and conduction velocity, and a decrease in mean conduction velocity in the inflected population, might best be explained by an increase in the relative proportions of myelinated nociceptors. Of greatest interest was the finding that in both NGF overexpressers and wild-type mice, the correlation between mechanical threshold and presence or absence of an inflection on the somal spike is not apparent by P5.

Animals↗

PubMed2000

Central nervous system leiomyosarcoma in patients with acquired immunodeficiency syndrome. Report of two cases.

Leiomyosarcomas (LMSs) of the central nervous system are extremely rare; however, they are becoming more prevalent in immunocompromised patients. The authors present the cases of two patients with acquired immunodeficiency syndrome: one with LMS of the thoracic vertebral body and the other with LMS originating from the region of the cavernous sinus. The epidemiological and histological characteristics of LMS and its association with latent Epstein-Barr virus are discussed, as well as the treatments for this neoplasm.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Peripheral specification of Ia synaptic input to motoneurons innervating foreign target muscles.

Muscle sensory neurons, called Ia afferents, make monosynaptic connections with functionally related sets of motoneurons in the spinal cord. Previous work has suggested that peripheral target muscles play a major role in determining the central connections of Ia afferents with motoneurons. Here, we ask whether motoneurons can also be influenced by their target muscles in terms of the monosynaptic input they receive from Ia afferents, by transplanting thoracic motoneurons into the lumbosacral spinal cord so that they innervate foreign muscles. Three or four segments of thoracic neural tube from stage 14-15 chicken embryos were transplanted to the lumbosacral region of stage 16-17 embryos, and electrophysiological recordings were made from transplanted motoneurons after the embryos had reached stage 38-40. Transplanted thoracic motoneurons innervated limb muscles and received monosynaptic inputs from Ia afferents. These connections were not random: Most of the connections were formed between Ia afferents and motoneurons projecting to the same muscle (homonymous connections). Few aberrant connections were found although the anatomical distribution of afferents in the transplant indicated that they had ample opportunity to contact inappropriate motoneurons. We conclude that although peripheral target cues are not sufficient to respecify an already committed motoneuron (turn a thoracic motoneuron into a lumbosacral motoneuron), they do provide sufficient information for Ia afferent input to be functionally correct.

Afferent Pathways↗

PubMed1999

Brain stem blood flow, pupillary response, and outcome in patients with severe head injuries.

OBJECTIVE: Acute pupillary dilation in a head-injured patient is a neurological emergency. Pupil dilation is thought to be the result of uncal herniation causing mechanical compression of the IIIrd cranial nerve and subsequent brain stem compromise. However, not all patients with herniation have fixed and dilated pupils, and not all patients with nonreactive, enlarged pupils have uncal herniation. Therefore, we have tested an alternative hypothesis that a decrease in brain stem blood flow (BBF) is a more frequent cause of mydriasis and brain stem symptomatology after severe head injury. We determined the relation of BBF to outcome and pupillary response in patients with severe head injuries. METHODS: One hundred sixty-two patients with a Glasgow Coma Scale score of 8 or less underwent stable xenon computed tomographic blood flow determination at the level of the superior colliculus, and this blood flow was correlated with pupillary features, intracranial pressure, computed tomographic scan pathology, and outcome. RESULTS: A BBF of less than 40 ml/100 g/min was significantly associated with poor outcome (P < 0.009). In patients with bilaterally nonreactive pupils, the BBF was 30.5+/-16.8 ml/100 g/min, and in those with normally reactive pupils, the BBF was 43.8+/-18.7 ml/100 g/min (P < 0.001). Intracranial pressure and the presence of a brain stem lesion observed on the computed tomographic scan did not correlate with BBF, pupillary size, or reactivity. Unfavorable outcome at 12 months was directly related to age (P = 0.062) and inversely related to pupillary responsiveness (P = 0.0006), pupil size (P = 0.005), and BBF of less than 40 ml/100 g/min (P = 0.009). CONCLUSION: These findings suggest that pupillary dilation is associated with decreased BBF and that ischemia, rather than mechanical compression of the IIIrd cranial nerve, is an important causal factor. More important, pupil dilation may be an indicator of ischemia of the brain stem. If cerebral blood flow and cerebral perfusion pressure can be rapidly restored in the patient with severe head injury who has dilated pupils, the prognosis may be good.

Adolescent↗

PubMed1999

Intracranial extension of an eccrine porocarcinoma. Case report and review of the literature.

Eccrine porocarcinoma is a rare malignant tumor of the true sweat gland. It commonly presents in the lower extremities with lymphatic metastasis. The authors describe the clinical presentation, radiographic evidence, operative discoveries, and pathological findings in a patient with an eccrine porocarcinoma involving the soft tissue of the occiput, which had eroded through the cranium. A review of the literature failed to reveal any other such case. The discussion includes the epidemiology, pathogenesis, treatment, and outcome of eccrine porocarcinomas. The six reported cases of scalp eccrine tumors are reviewed.

Acrospiroma↗

PubMed1998

Ependymomas: MIB-1 proliferation index and survival.

The biologic behavior of ependymomas is highly variable, and its correlation with histologic features is at best imprecise. This retrospective study attempted to correlate the malignant histologic characteristics of ependymomas with MIB-1 proliferation index and survival. Biopsy and resection specimens taken from 34 patients who received treatment 1972 to 1996 were histologically examined. The patients' ages range was 1 to 59 years. The histologic specimens were assessed for anaplastic features (necrosis, mitosis, vascular proliferation, cellular pleomorphism, and overlapping of nuclei) and an MIB-1 (Ki-67 antigen) proliferation index was also determined. The overall median MIB-1 proliferation index was 7.8% (range 0.1 - 62.5%). An MIB-1 of 20% was significant for a decrease in survival (RR = 5.7) (p = 0.0013). The median MIB-1 for patients < 20 years old was 20.6% with range (0.1, 43%), while that for patients > 20 years was 5.1% (range 0.2, 9.4%) (KW p = 0.055). Three of 5 histological features evaluated were significantly associated with outcome: > 5 mitotic figures per high-power field, necrosis, and vascular proliferation, but not nuclear overlap or pleomorphism. All pathologic factors except pleomorphism were significantly related to the MIB-1 proliferation index. In brief, our data support the association of poor prognoses in ependymomas with young age, the presence of three to four anaplastic histologic features, and an MIB-1 proliferation index > 20%.

Adolescent↗

PubMed1996

Evaluation of a regional oxygen saturation catheter for monitoring SjvO2 in head injured patients.

OBJECTIVES: Monitoring jugular venous oxygen saturation (SjvO2) has been useful for the early identification and treatment of cerebral ischemia in patients with severe head injury. However, the catheters that have been used for this purpose have not performed optimally. The purpose of this study was to evaluate the performance of a new regional oxygen saturation catheter for monitoring SjvO2. METHODS: Eighteen regional oxygen saturation catheters, 4-Fr in diameter (Baxter Healthcare Corporation, Edward Critical Care), were used in this study. Each catheter was inserted percutaneously into the dominant jugular vein and the catheter's tip position in the jugular bulb was verified by radiograph. The catheter was calibrated in vitro prior to insertion using the optic calibrator provided by the manufacturer. The catheter was recalibrated every 8 to 12 hours by comparing the oxygen saturation value from the catheter with that measured by a co-oximeter in a blood sample drawn through the catheter. RESULTS: In vitro calibration using the optic calibrator was not always successful. Five catheters could not be calibrated. The remaining 13 catheters could all be calibrated, but only 9 provided a value that was within 4% of the oxygen saturation derived from the blood sample. After the first in vivo calibration, the correlation between the catheter and the blood sample values was improved. A total of 196 comparisons were made. The median, 25th, and 75th quartile differences between the catheter and the blood sample measurement of SjvO2 were 0.00, -1.15, and 1.25%, respectively. Using longitudinal data regression, the overall slope of the regression between the catheter and blood values was 0.997 (p = 0.001). CONCLUSIONS: The new regional oxygen saturation catheter provided reliable measurement of SjvO2 83% of the time when the signal quality index was < or = 3, and may be useful for continuous monitoring of SjvO2.

Blood Gas Analysis↗

PubMed1996

Evaluation of a carbohydrate-free diet for patients with severe head injury.

Hyperglycemia, which may be caused or exacerbated by conventional diets, may worsen the neurological outcome from severe head injury, especially if secondary ischemic insults occur. The purpose of this study was to evaluate an experimental diet intended to replace systemic caloric and protein requirements without producing hyperglycemia. In initial studies in the laboratory, 5 experimental diets were employed in a middle cerebral artery temporary occlusion model. The effects of the diets on blood biochemistry and on infarction volume were compared in fasted animals and in animals fed a control diet. Animals fed the experimental diets had a significantly lower preischemia blood glucose concentration, a higher blood concentration of ketone bodies, and a smaller infarct volume than the animals fed a control diet. One diet chosen from the laboratory study was then evaluated in a clinical study as a randomized, open-label trial. Twenty severely head-injured patients were randomly assigned to be fed the experimental diet, EN-9305, or the control diet, Osmolyte HN, for the first 2 weeks after injury. Both treatment groups had similar blood glucose concentrations, averaging 6.33 +/- 0.21 mumol/mL (114 +/- 4 mg/dL), on day 1 prior to starting the assigned diet. Blood glucose concentration increased in the control diet group to a peak of 8.37 +/- 0.94 mumol/mL (151 +/- 17 mg/dL) on day 7 as the infusion rate of the diet was increased to the final rate. In the experimental diet group, the blood glucose concentration remained unchanged from fasting levels as the diet was advanced. Blood lactate concentration was lower, and blood ketone body concentrations were higher in the patients fed the experimental diet. Urinary nitrogen balance was better in the experimental diet group, but measures of visceral protein sparing, including serum albumin, plasma retinol binding protein, and total lymphocyte count, were not significantly different in the 2 treatment groups. Measures of cerebral anaerobic metabolism, including CSF lactate concentration and cerebral lactate production, were not significantly different in the 2 treatment groups. These studies suggest that a carbohydrate-free diet such as EN-9305 might have advantages for patients with severe head injury by replacing systemic caloric and protein requirements without producing hyperglycemia.

Adult↗

PubMed1996

Intrasellar presentation of a cystic optic chiasm fibrillary astrocytoma.

The unusual radiologic presentation of an optic chiasm fibrillary astrocytoma extending through and expanding the diaphragma sellae in an 8-year-old male is described. The child presented with decreased vision in the right eye. Magnetic resonance imaging demonstrated a cystic intra- and suprasellar mass, isointense on T1WI and hyperintense on T2WI, that enhanced with contrast. There was no radiographic involvement of the pituitary or hypothalamus. The optic chiasm could not be seen. The tumor mass was believed to be a craniopharyngioma and was partially removed by a transsphenoidal approach. Frozen section was not consistent with craniopharyngioma. Histochemical stains were positive for glial fibrillary acidic protein and S100 protein and nonreactive for EMA or actin. Electron microscopy showed abundant cellular processes and cytoplasmic filaments within the cells. A diagnosis of intrasellar fibrillary astrocytoma, probably arising from the optic chiasm, was made.

Astrocytoma↗

PubMed1994

Prognostic significance of bromodeoxyuridine labeling in primary and recurrent glioblastoma multiforme.

To evaluate the prognostic significance of bromodeoxyuridine (BUdR) labeling index (LI) and to estimate tumor proliferative potential, BUdR LI was examined in 98 patients having a primary diagnosis of glioblastoma multiforme (GBM); 55 underwent infusion of 200 mg/m2 of BUdR at the time of the primary resection and 49 underwent infusion at the time of the second resection. The tumors of six patients were labeled at both operations. The tumor specimens were stained with hematoxylin and eosin for histopathology and by immunohistochemical methods to determine the ratio of labeled to unlabeled cells, i.e., BUdR LI. The median BUdR LIs for the primary and recurrent GBM were significantly different at 6.8 and 2.6%, respectively (P < 0.0001). However, there was no significant association between BUdR LI at the first or second operation and survival (log rank, P = 0.12; Cox regression analysis, P = 0.91; log rank, P = 0.55; Cox regression analysis, P = 0.17, respectively). Patients who underwent a second operation within 10 months of the first operation had a lower BUdR LI than did patients with a longer interval between procedures (P = 0.078; Spearman rank correlation, 0.26). The aggressive biological behavior of GBM is dependent on complex cellular kinetics, not simply on the number of cells within the S phase of the cell cycle. Caution should be used when determining prognosis with BUdR LI in GBM.

Adult↗

PubMed1994

Cerebral metabolism.

An intricate relationship normally exists between cerebral metabolism and energy substrate supply because of the brain's high substrate demand and limited storage capacity. In head-injured patients, this balance can be disrupted. The brain parenchyma directly involved by the injury is hypometabolic in respect to glucose and oxygen, whereas peri-injury tissue may have an elevated metabolic rate.

Brain↗

PubMed1993

Regulation of myelinated nociceptor function by nerve growth factor in neonatal and adult rats.

The role of nerve growth factor (NGF) as a survival factor for sensory neurons during embryonic life has been well documented. Here we examine the actions of NGF or antisera against NGF (anti-NGF) on physiologically identified sensory neurons with myelinated axons later in life, after the dependence on NGF for survival ends. We find that the effects of NGF and anti-NGF are specific for sensory neurons which are nociceptors. Treatments were found to affect the biophysical properties, the development, or the physiological function of myelinated nociceptors. They also affect the animal's behavioral response to noxious stimulation, depending upon when the treatments were given: neonatally, from 2-5 weeks of age, or chronically, beginning at birth. Thus, we find that the actions of NGF are specific for nociceptors but that the function of this neurotrophic factor changes according to the developmental age of the animal.

Animals↗

PubMed1993

CT-guided stereotactic biopsy of nonenhancing brain lesions.

The majority of computer tomography stereotactic biopsy lesions enhance after administration of intravenous contrast, whereas patients with nonenhancing lesions are often followed conservatively or undergo craniotomies. There are few studies showing the effectiveness of stereotactic biopsies of nonenhancing cerebral lesions. Stereotactic biopsies were performed on 19 patients with lesions that did not enhance on CT after intravenous contrast. Pathological diagnoses were made in 90% (17/19) of patients. Four HIV-positive patients had progressive multifocal leukoencephalopathy, 11 patients had gliomas (4 astrocytomas, 6 anaplastic astrocytomas, and 1 ganglioma), 1 had multiple sclerosis, and 1 had herpes encephalitis. In 2 patients multiple biopsies revealed only gliosis. There was no morbidity or mortality. Stereotactic biopsies for nonenhancing brain lesions have a high diagnostic yield and can favorably alter the treatment course.

AIDS Dementia Complex↗

PubMed1993

Nerve growth factor-induced hyperalgesia in the neonatal and adult rat.

Recently, we have shown that the interaction between NGF and sensory neurons in early postnatal periods is restricted to nociceptive afferents (Ritter et al., 1991; Lewin et al., 1992a; Ritter and Mendell, 1992). Here we show that administration of excess NGF to neonatal or mature animals can lead to a profound behavioral hyperalgesia. Neonatal NGF treatment (postnatal day 0-14) resulted in a profound mechanical hyperalgesia that persisted until the animals had reached maturity (6 weeks of age). This hyperalgesia could be explained by an NGF-mediated sensitization of A delta nociceptive afferents to mechanical stimuli. This peripheral sensitization wore off with a time course similar to that of the behavior hyperalgesia. Treatment of animals from the second postnatal week until 5 weeks of age (juveniles) led to a very similar behavioral hyperalgesia; however, there was no corresponding sensitization of A delta nociceptors to mechanical stimuli. Finally, one group of adult animals (5 weeks old) was treated daily with single injections of NGF for between 1 and 4 d. Within 24 hr after the first NGF injection these animals developed a mechanical hyperalgesia of the same magnitude seen after neonatal and juvenile NGF treatments. No sensitization of A delta nociceptive afferents was observed in these animals. In addition to the mechanical hyperalgesia, the animals also developed a heat hyperalgesia after one injection of NGF. The heat hyperalgesia was apparent within 15 min after the injection; however, signs of mechanical hyperalgesia were not seen until 6 hr after the injection. In conclusion, it appears that the NGF-induced mechanical hyperalgesia is brought about by different mechanisms in neonatal and adult rats. Furthermore, in adult animals the NGF-induced mechanical and heat hyperalgesia also appear to be attributable to two different mechanisms. The mechanical hyperalgesia may be due to central changes (see Lewin et al., 1992b), whereas the heat hyperalgesia is likely to result at least in part from the sensitization of peripheral receptors to heat.

Aging↗

PubMed1992

Characterization of solubilized opioid receptors: reconstitution and uncoupling of guanine nucleotide-sensitive agonist binding.

Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-(CHAPS). High concentrations of NaCl (0.5-1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane-bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High-affinity binding of mu, delta, and kappa agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, mu-agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin ([3H]DAGO) binding to guanosine 5'-O-(thiotriphosphate) (GTP gamma S). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTP gamma S was observed, whereas membrane-bound receptors were uncoupled only partially. Treatment with GTP gamma S, at concentrations that uncoupled the mu receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗