A System for Implanting Laboratory Mice with Light-Activated Microtransponders

The mouse is the most commonly used laboratory animal, accounting for up to 80% of all mammals used in research studies. Because rodents generally are group-housed, an efficient system of uniquely identifying individual animals for use in research studies, breeding, and proper colony management is required. Several temporary and permanent methods (for example, ear punching and toe clipping) are available for labeling research mice and other small animals, each with advantages and disadvantages. This report describes a new radiofrequency identification tagging method that uses 500-µm, light-activated microtransponders implanted subcutaneously into the ear or tail of mice. The preferred location for implanting is in the side of the tail, because implantation at this site was simple to perform and was associated with shorter implantation times (average, 53 versus 325 s) and a higher success rate (98% versus 50%) compared with the ear. The main benefits of using light-activated microtransponders over other identification methods, including other radiofrequency identification tags, is their small size, which minimizes stress to the animals during implantation and low cost due to their one-piece (monolithic) design. In addition, the implantation procedure uses a custom-designed 21-gauge needle injector and does not require anesthetization of the mice. We conclude that this method allows improved identification and management of laboratory mice.Abbreviation: RFID, radiofrequency identification; ID, serial numberLaboratory mice play an important role in basic, biomedical, and mammalian research. Inbred and mutant mice are accepted universally as the primary model for analyzing and understanding inherited human disorders.¹⁷ Over 75 million laboratory mice are used worldwide annually, including 25 million in the United States. Much of this use is in biomedical research and pharmaceutical drug development; therefore mice play a key part in improving healthcare for people around the world. Mice are social animals, and group-housing, at recommended densities, generally is considered to reduce stress in rodents.^6,9,16 However, this housing method requires an efficient system of uniquely identifying individual animals. Despite this need, our ability to identify and keep track of laboratory mice is limited. The most widely used methods for permanently identifying rodents—ear punching, ear tagging, and toe clipping—are relatively primitive. The number of possible identification numbers is limited with ear and toe clipping, thereby complicating breeding programs. In addition, these methods are potentially stressful for animals and do not foster ‘cradle-to-grave’ tracking. Compared with clipping methods, ear tags offer a greater number of identification codes and are simple to apply; however, they can cause irritation and are often subject to self-removal.^2,19 Each of these methods may lead to errors associated with misreading by technicians, and, in some cases, lead to repetition of partial or full experiments, a situation that is both costly and labor-intensive.Recently, radiofrequency identification transponders (RFID tags), which are implanted subcutaneously, have been used to label mice, given that these tags address several of the problems associated with nonelectronic methods. An RFID tag typically consists of electronic circuitry and a solenoid antenna enclosed in a glass capsule. The transponders transmit unique alphanumeric codes that are easily distinguished from each other by a dedicated reading device. The typical size of an RFID transponder capsule is 13 × 2 mm, although somewhat smaller RFID tags (6 × 1 mm) are available also. RFID tags are manufactured and distributed by several vendors.^1,7,13 However, the current tags have not been cost-effective for routine identification of laboratory mice and, therefore, their use has primarily been limited to high-value animals used in specialized studies.The purpose of the present report is to describe the use of a new type of RFID tag for identifying laboratory mice. It is based on a very small microtransponder that transmits a numerical serial code when activated by laser light. Microtransponders have been used previously in cell-based¹² and multiplex DNA¹¹ assays and as a means of tracking ants in behavioral studies.¹⁵ The main benefits of these devices over other RFID transponders is their small size, which minimizes stress to the animals during implantation, and low cost, due to their monolithic design. The microtransponder surface is silicon dioxide, similar to capsule-type implantable tags, a feature that is important for biocompatibility. The RFID tag is part of a system with a dedicated reader, software, and specialized injection device for implanting mice. These components combine to provide a system that simplifies identification individual mouse and tracking and, if widely adopted, can lead to improvements in animal welfare.     

Expression of serotonergic receptors in psoriatic skin

Psoriasis appears to be influenced by stress, which causes release of adrenal hormones. Serotonin, or hormonal actions on serotonin and serotonin receptors, may have a role in psoriasis. Distribution of serotonin receptors was studied in involved and noninvolved skin in patients with psoriasis and compared to normal skin, by using immunohistochemistry and antibodies to 5-HT1A, 5-HT2A and 5-HT3 receptors (R). There was a decreased (P<0.001) number of 5-HT1AR positive cells, the majority being tryptase positive, in involved and noninvolved psoriatic papillary dermis, compared to normal skin. 5-HTlAR expression was also found in the upper part of the epidermis, on vessel walls and on melanocytes. 5-HT2AR expressing papillary mononuclear cells, CD3 positive, were increased (P<0.001 and P<0.01, respectively) in involved and noninvolved psoriatic skin, compared to normal skin, an increase (P<0.01) also being found in the involved compared to noninvolved skin. Expression of 5-HT3R could be found in the basal epidermal layer of noninvolved but not in the involved skin of psoriasis, where it was only found in the acrosyringium. The present findings are compatible with the 5-HT1A and 5-HT2A receptors having antagonistic functions, and raise the possibility of using receptor specific drugs in the treatment of psoriasis.     

Effects of acute and chronic administration of Leu-enkephalin on cultured serotonergic neurons: evidence for opioids as inhibitory neuronal growth factors

Leu-enkephalin, at concentrations between 18 microM and 1.8 pM, was administered in a single or daily dose to dissociated mesencephalic raphe cell cultures maintained for 3 or 5 days. Daily administration of Leu-enkephalin produced an inhibition of high affinity uptake of [3H]5-HT, a measure of serotonergic process outgrowth in cultures of fetal neurons. This inhibition was maximal at a dose of 18 nM in both 3 (59%, P less than 0.05)- and 5 (38%, P less than 0.05)-day cultures. The expression of uptake was consistently lower in 5-day cultures than in 3-day cultures at all concentrations tested. In marked contrast, a single dose of Leu-enkephalin at the time of plating stimulated uptake in 3- and 5-day cultures. Maximal stimulation was observed at 180 nM for both 3 (191%, P less than 0.05)- and 5 (140%, P less than 0.05)-day cultures. The results obtained after a single dose of the opioid may reflect a paradoxical stimulation probably due to a rebound mechanism of receptors since co-administration of bacitracin (0.5 mg/ml), an aminopeptidase inhibitor, resulted in inhibition of the uptake expression. Together these results indicate that Leu-enkephalin can function as an inhibitory regulatory growth factor for neuronal cultures when constant exposure to this opioid is maintained over time.     

Effect of cupric ions on the initiation protein synthesis rate in the human endometrium

The effect of cupric ions on the initiation protein synthesis rate of the human endometrium was studied. Addition of copper to the complete ribosomal system decreased the binding of [³H]Met-tRNA(i) to the isolated ribosomes with a plateau at about 70% inhibition with concentrations higher than 150 μM.

The initiation activity was GTP-dependent with a maximum at 2 mM. This activity was very rapid, requiring 5 min to complete the reaction. Incubation of isolated initiation factors with copper (300 μM) inhibited the formation of the ternary complex. When the complete system was reconstituted with salt-washed ribosomes after ternary complex formation, no significant change on the inhibition pattern was observed. Addition of initiation factors to 5-min preincubated salt-washed ribosomes with 300 μM copper, after the elimination of excess copper, induced only a 12% decrease on Met-tRNA(i) binding. This effect was not modified by the presence of Sparsomycin, an elongation inhibitor. It was concluded that copper interferes with the initiation process, probably at the ternary complex formation level.     

Colchicine-induced cytoskeletal collapse and apoptosis in N-18 neuroblastoma cultures is rapidly reversed by applied S-100β

Brain connections depend on a stable association between dendrites and axons whose cytoskeleton is stabilized by the proteins MAP-2 and tau, respectively. The glial protein S-100β inhibits the phosphorylation by PKC of these two microtubule-associated proteins. In order to determine if exogenous S-100β can directly influence the cytoskeleton of living cells, cultures of N-18 cells (neuroblastoma clonal cell line) are treated for 30 min in serum-free medium with 10⁻⁶ M colchicine. In normal media, colchicine induces a rapid retraction of processes, membrane blebbing, nuclear collapse, and cell death. The observed cellular changes, due to cytoskeletal collapse after exposure to colchicine, are similar and consistent with the loss of processes and cytoplasmic blebbing seen in cells undergoing apoptosis. The addition of 20 ng/ml of S-100β after the initial 30-min exposure to colchicine prevents apoptosis, nuclear collapse and induces the regrowth of retracted processes. Cells were treated with the Hoechst Stain, a fluorescent marker that binds to nuclear material, to determine the occurrence of apoptosis in our cultures. In our control cultures, receiving no drugs, we found that 15.1% of the cells were apoptotic. When colchicine was added to the culture medium we found that 31.6% of the cells became apoptotic. However, when colchicine was followed by exposure to S-100β we found that only 5.4% of the cells were apoptotic. Our results suggest that extracellular application of the glial protein S-100β is sufficient to reverse colchicine-induced cytoskeletal collapse and prevent the resultant apoptosis of the cells. The increased levels of S-100β seen after brain injury and in certain neurological and psychiatric disorders may be considered as beneficial for brain recovery.     

Age related changes in NGF, EGF and protease in the granular convoluted tubules of the mouse submandibular gland. A morphological and immunocytochemical study

Granular convoluted tubule (GCT) cells in the submandibular glands of male mice were studied at 1.5, 6, 12 and 29 months of age by morphometric and immunocytochemical means. The relative proportion of GCT's was established by determining a tubule to acini (T/A) ratio. The T/A ratios were significantly increased between 1.5 months (0.76) and 6 months (1.34), and between 6 months and 12 months (1.96). However, a marked decrease was found at 29 months (0.59). The functional capacity of GCT cells was estimated by immunocytochemical staining for nerve growth factor (NGF), epidermal growth factor (EGF), protease and renin. The number of GCT cells immunoreactive for NGF, EGF and protease increased between 1.5 months and 6 months. No further change was noted at 12 months. However, once again at 29 months a decrease occurred in a number of cells reactive for all three substances. Renin was not detected in any cell at any age.