PubMed Health⌕ Search

Biomedical subjects

D Durham

Publications and source records attributed to D Durham.

At least 19 recordsLinked to original sources
PubMed2001

An integrated process for measuring the physicochemical properties of drug candidates in a preclinical discovery environment.

Automated log P, pK(a), solubility, and chemical stability systems comprise an integrated process that provides early stage physicochemical property data to the discovery research organization. Capillary electrophoresis (CE) techniques are used to experimentally determine pK(a) and log P. Solubility is determined using a quasi-equilibrium approach employing sample quantitation by flow injection analysis with ultraviolet (UV) detection at 256 nm. Chemical stability is assessed by challenging compounds with pH 2, pH 7, pH 12, and 3% hydrogen peroxide solutions overnight, and comparing the chromatographic profiles of the stability challenged solutions to that of a freshly prepared control. Validation of the log P method using a set of drug-like compounds demonstrates that the method yields log P values within +/-0.5 units of literature values. The log P method is valid over the range -0.5-5.0, and the technique is compatible with acidic, neutral, and basic compounds. The pK(a) technique yields results within +/-0.2 units of corresponding values obtained by potentiometric titration over a pK(a) range of 2 to 12. Solubility is reported in a 3-60 microg/mL range, and the results are generally within 20% of values measured by equilibrium solubility techniques. The current level of automation supports the measurement of the physicochemical properties of 100 compounds per week. Physicochemical property data for approximately 2000 compounds have been generated to date.

Automation↗
PubMed2001

Effects of conductive hearing loss on gerbil central auditory system activity in silence.

Animal models of conductive hearing loss (CHL) show altered structure and function in the central auditory system (CAS), particularly following unilateral deprivation. Assessment of neuronal activity as measured by 2-deoxyglucose (2-DG) uptake following CHL has been reported by two groups of investigators, with different findings. Woolf and colleagues [Brain Res. 274 (1983) 119] found that 2-DG uptake increased in the cochlear nucleus ipsilateral to the CHL, while Tucci et al. [Laryngoscope 109 (1999) 1359] found a decrease in 2-DG uptake in the ipsilateral cochlear nucleus. One significant difference between the protocols in the two studies was that, in the first study, animals were maintained in silence following 2-DG injection, whereas in the Tucci et al. study, animals were exposed to sound. The current study was designed to replicate the protocol used by Woolf et al. Young adult gerbils underwent unilateral malleus removal with bilateral canal ligation (n=6) or a sham procedure (n=7) 48 h prior to 2-DG administration and sacrifice. Optical density measurements were made from CAS nuclei. 2-DG uptake decreased in the ipsilateral cochlear nucleus and contralateral inferior colliculus, and in nuclei of the superior olivary complex bilaterally, supporting the finding that CHL is associated with a decrease in CAS neuronal activity.

Acoustic Stimulation↗
PubMed2000

Rapid method for estimating the octanol--water partition coefficient (log P ow) by microemulsion electrokinetic chromatography.

Several surfactant systems were evaluated based on their system constants determined by the solvation parameter model for the design of a surrogate chromatographic model for the rapid estimation of octanol-water partition coefficient (log Pow) by microemulsion electrokinetic chromatography. The system constant ratios responsible for the log Pow partition system are (nearly) the same as those for the microemulsion system containing sodium dodecyl sulfate (1.4% w/v), butan-1-ol (8% v/v) and heptane (1.2% v/v). Neutral and basic compounds are analyzed using a fused-silica capillary column with a 50 mM sodium phosphate-sodium borate (3:2) buffer at pH 10. Weakly acid compounds require the use of sulfonated silica capillary column and a 50 mM sodium phosphate buffer at pH 3. For 29 varied neutral and weakly basic compounds the average error between log Pow estimated using MEEKC and literature values was +/-0.12 over a log Pow range from 0.3 to 5.8.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2000

Central nervous system plasticity during hair cell loss and regeneration.

Following cochlear ablation, auditory neurons in the central nervous system (CNS) undergo alterations in morphology and function, including neuronal cell death. The trigger for these CNS changes is the abrupt cessation of afferent input via eighth nerve fiber activity. Gentamicin can cause ototoxic damage to cochlear hair cells responsible for high frequency hearing, which seems likely to cause a frequency-specific loss of input into the CNS. In birds, these hair cells can regenerate, presumably restoring input into the CNS. This review summarizes current knowledge of how CNS auditory neurons respond to this transient, frequency-specific loss of cochlear function. A single systemic injection of a high dose of gentamicin results in the complete loss of high frequency hair cells by 5 days, followed by the regeneration of new hair cells. Both hair cell-specific functional measures and estimates of CNS afferent activity suggest that newly regenerated hair cells restore afferent input to brainstem auditory neurons. Frequency-specific neuronal cell death and shrinkage occur following gentamicin damage to hair cells, with an unexpected recovery of neuronal cell number at longer survival times. A newly-developed method for topical, unilateral gentamicin application will allow future studies to compare neuronal changes within a given animal.

Animals↗
PubMed2000

Neuronal death, not axonal degeneration, results in significant gliosis within the cochlear nucleus of adult chickens.

Injury to the central nervous system initiates a series of events that leads to neuronal cell death and glial activation. Astrocytes respond to damage and disease by becoming hyperplastic and hypertrophied. This 'reactive gliosis' is also accompanied by the upregulation of the intermediate filament protein glial fibrillary acidic protein, the release of growth factors and the formation of the glial scar. However, the signaling cascades which regulate these events, and the molecular mechanisms that give rise to this diverse response, have not been fully elucidated. For example, the role played by degenerating neurons vs. degenerating axons in the activation of astrocytes remains to be determined. To investigate the influence of neuronal cell death vs. axonal degeneration on gliosis, the current study examines the astrocyte response to cochlea removal in two different breeds of adult chickens, one of which exhibits neuronal cell death within the brainstem nucleus magnocellularis (NM) following the lesion and one which does not. Our results indicate that degeneration of NM neurons leads to large increases in both glial proliferation and hypertrophy, while eighth nerve degeneration without NM cell death results in very small increases in glial proliferation.

Animals↗
PubMed1999

Breed differences in deafferentation-induced neuronal cell death and shrinkage in chick cochlear nucleus.

Removal of functional presynaptic input can result in a variety of changes in postsynaptic neurons in the central nervous system, including altered metabolism, changes in neuronal cell size, and even death of the postsynaptic cell. Age-dependent neuronal cell death and shrinkage has been documented in second order auditory neurons in the chick brainstem (nucleus magnocellularis, NM) following cochlea removal (Born and Rubel, 1985. J. Comp. Neurol. 231, 435-445). Here we examined whether the extent of neuronal cell death and shrinkage is also breed-dependent. We performed unilateral cochlea removal on both hatchling and adult birds of either a broiler breed (Arbor Acres Cross) or egg layer breed (Hy-Line, H and N) and killed birds one week later. Changes in neuronal cell number and cross sectional area were determined from Nissl-stained sections. We observed 25% neuronal cell loss and a 15-20% decrease in neuronal cross sectional area after cochlea removal in either broiler or egg layer hatchling birds. In adult birds, however, neuronal cell loss is breed-dependent. Adult egg layer birds lose an average of 37% of NM neurons after cochlea removal, while adult broiler birds show no cell loss. In both breeds of adult birds, cochlea removal results in a 20% decrease in neuronal cross sectional area. These results suggest that analysis of differences between breeds as well as ages of birds will prove fruitful in determining how afferent input controls neuronal survival and metabolism.

Animals↗
PubMed1999

Tonotopic changes in 2-deoxyglucose activity in chick cochlear nucleus during hair cell loss and regeneration.

Following cochlear ablation, auditory neurons in the central nervous system (CNS) undergo alterations in morphology and function, including neuronal cell death. The trigger for these CNS changes is the abrupt cessation of eighth nerve fiber activity. Gentamicin can cause ototoxic damage to cochlear hair cells responsible for high frequency hearing. In birds, these hair cells can regenerate. Therefore, gentamicin causes a partial, yet reversible insult to the ear. It is not known how this partial hair cell damage affects excitatory input to the cochlear nucleus. We examined chick cochlear nucleus activity during hair cell loss and regeneration by measuring 2-deoxyglucose (2DG) uptake. Normal animals showed a rostral to caudal gradient of 2DG activity, with higher activity in caudal regions. When hair cells are damaged (2, 5 days), 2DG uptake is decreased in cochlear nucleus. When hair cells regenerate (9, 16, 28 days), 2DG uptake returns to control levels. This decrease and subsequent return of activity only occurs in the rostral, high frequency region of the cochlear nucleus. No changes are seen in the caudal, low frequency region. These results suggest that changes in activity of cochlear nucleus occur at a similar time course to anatomical changes in the cochlea.

Acoustic Stimulation↗
PubMed1999

Conductive hearing loss results in a decrease in central auditory system activity in the young gerbil.

OBJECTIVES/HYPOTHESIS: The impact of childhood conductive HL (CHL) on development of auditory function has long been debated. The present study was conducted to define and compare the consequences of CHL and cochlear ablation (CA) in young and adult animals, using 2-deoxyglucose (2-DG) uptake as a measure of metabolic activity. It was hypothesized that, for both ages, CHL would result in a decrease in activity in the major ascending central auditory system pathway of the manipulated ear, but that this decrease would be significantly less than that observed with CA. STUDY DESIGN: Sham-controlled study of metabolic effects of CHL during sound stimulation. METHODS: Gerbils (aged 21 days or adult), underwent malleus removal, CA, or a sham procedure. Young animals survived either 48 hours or 3 weeks; adults survived 3 weeks. Each age/survival CHL group contained eight animals; otherwise, each group (CA and sham) contained five animals, for a total number of 54. At the appropriate survival time, animals were given an intracardiac injection of 14C-2-DG, and sacrificed under anesthesia after 45 minutes of exposure to normal laboratory sounds. Tissue sections were prepared for exposure to x-ray film for optical density measurements, and alternate sections stained for identification of nuclei. Measurements from auditory nuclei of experimental animals were corrected against an unaffected control area (abducens nucleus) and compared with measurements taken from animals in the sham group. Auditory evoked potential thresholds to both air- and bone-conducted stimuli were obtained in a second group of neonatal and adult animals. RESULTS: Both CHL and CA resulted in a marked decrease in 2-DG uptake in the major ascending projection of the manipulated ear, in both the neonatal and adult animals. In young animals, effects of CHL and CA were similar. Effects of CHL in adult animals were less marked and significantly different from either effects of CHL in young animals or effects of CA in adult animals. HL following malleus removal only was purely conductive and ranged from 38 to 55 dB across frequency. CONCLUSIONS: Results suggest that, particularly in young animals, a unilateral CHL may have profound effects on metabolic activity in the central auditory system.

Animals↗
PubMed1998

Round window administration of gentamicin: a new method for the study of ototoxicity of cochlear hair cells.

Damage to inner ear sensory hair cells after systemic administration of ototoxic drugs has been documented in humans and animals. Birds have the ability to regenerate new hair cells to replace those damaged by drugs or noise. Unfortunately, the systemic administration of gentamicin damages both ears in a variable fashion with potentially confounding systemic drug effects. We developed a method of direct application of gentamicin to one cochlea of hatchling chickens, allowing the other ear to serve as a within-animal control. We tested variables including the vehicle for application, location of application, dosage, and duration of gentamicin exposure. After 5 or 28 days survival, the percent length damage to the cochlea and regeneration of hair cells was evaluated using scanning electron microscopy. Controls consisted of the opposite unexposed cochlea and additional animals which received saline instead of gentamicin. Excellent damage was achieved using gentamicin-soaked Gelfoam pledgets applied to the round window membrane. The percent length damage could be varied from 15 to 100% by changing the dosage of gentamicin, with exposures as short as 30 min. No damage was observed in control animals. Regeneration of hair cells was observed in both the base and apex by 28 days survival.

Animals↗
PubMed1998

Evidence for loss and recovery of chick brainstem auditory neurons during gentamicin-induced cochlear damage and regeneration.

It is well documented that damage to the chick cochlea caused by acoustic overstimulation or ototoxic drugs is reversible. Second-order auditory neurons in nucleus magnocellularis (NM) are sensitive to changes in input from the cochlea. However, few experiments studying changes in NM during cochlear hair cell loss and regeneration have been reported. Chicks were given a single systemic dose of gentamicin, which results in maximal hair cell loss in the base of the cochlea after 5 days. Many new hair cells are present by 9 days. These new hair cells are mature but not completely recovered in organization by 70 days. We counted neurons in Nissl-stained sections of the brainstem within specific tonotopic regions of NM, comparing absolute cell number between gentamicin- and saline-treated animals at both short and long survival times. Our data suggest that neuronal number in rostral NM parallels hair cell number in the base of the cochlea. That is, after a single dose of gentamicin, we see a loss of both cochlear hair cells and NM neurons early, followed by a recovery of both cochlear hair cells and NM neurons later. These results suggest that neurons, like cochlear hair cells, can recover following gentamicin-induced damage.

Animals↗
PubMed1994

Rapid increase in mitochondrial volume in nucleus magnocellularis neurons following cochlea removal.

Second-order auditory neurons in nucleus magnocellularis (NM) of the chick brainstem undergo a series of rapid metabolic changes following unilateral cochlea removal, culminating in the death of 25% of NM neurons. Within hours of cochlea removal, ipsilateral NM neurons show marked increases in histochemical staining for the mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase. We investigated corresponding ultrastructural changes in NM neurons by preparing animals undergoing unilateral cochlea removal for transmission electron microscopy. We quantified changes in NM mitochondrial volume by stereological methods and qualitatively compared mitochondrial morphology between NM neurons destined to survive and those destined to die after cochlea removal. Within hours of cochlea removal, ipsilateral NM neurons show striking increases in mitochondrial volume (84% at 6 hours and 236% at 12 hours after cochlea removal compared to unoperated, control animals). At 2 week survival times, ipsilateral NM neurons contain fewer mitochondria than contralateral neurons. Surprisingly, anesthesia alone causes short-term increases in NM mitochondrial volume. Animals anesthetized with pentobarbital and ketamine and sacrificed 6 or 12 hours later showed a 45% increase in mitochondrial volume compared to previously unanesthetized animals. NM neurons destined to die within days of cochlea removal can be identified within several hours after deafferentation by the appearance of their ribosomes. We observed qualitative differences in mitochondrial morphology in dying neurons. Mitochondria in neurons destined to die consistently showed mitochondrial swelling and vacuolization indicative of metabolic dysfunction. Similar mitochondrial changes have been reported when mitochondria take up excess calcium. Ultrastructural changes in NM after cochlea removal display features of both programmed and pathological cell death, in which increased intracellular calcium is thought to play a role.

Anesthesia↗
PubMed1994

Increased deafferentation-induced cell death in chick brainstem auditory neurons following blockade of mitochondrial protein synthesis with chloramphenicol.

Second-order auditory neurons in nucleus magnocellularis (NM) of the chick brainstem undergo a series of rapid metabolic changes following unilateral cochlea removal, culminating in the death of 25% of NM neurons. Within hours of cochlea removal, ipsilateral NM neurons show marked increases in histochemical staining for the mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase (CO). We have shown previously in an ultrastructural study that these increases in oxidative capacity are mediated in part by a rapid increase in mitochondrial volume within deafferented neurons. In neurons that are destined to die as a result of deafferentation, mitochondria are smaller, stain poorly for CO, and often contain vacuoles associated with oxidative dysfunction. Our present set of experiments is designed to test the hypothesis that increases in oxidative metabolism are necessary for NM neuronal survival following removal of afferent input. We used chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor, to block the characteristic increase in CO activity and mitochondrial proliferation following cochlea removal. We then studied the effects of CAP on NM neuronal survival following deafferentation. When CAP was administered continuously at 600 mg/kg/d for 5 d following cochlea removal, deafferentation-induced neuronal death in NM was significantly increased from 22% to 36%. Higher-dose (1200 mg/kg/d) pulses of CAP were administered for the first 6, 12, or 24 hr following cochlea removal. After 5 d survival, greater increases in neuronal cell death were found in animals treated for the first 12 or 24 hr (65% neuronal death). CAP administration for the first 6 hr had no significant effect on neuronal survival. The effects of CAP on neuronal cell death in NM are not likely to be due to systemic effects of the drug, but instead to a specific change in mitochondrial function. Our results suggest that enhanced oxidative enzyme function plays an important role in the survival of NM neurons during the first 24 hr after deafferentation. The nature of the signal(s) eliciting mitochondrial enhancement and the means by which it influences NM survival are not known.

Afferent Pathways↗
PubMed1993

Altered malate dehydrogenase activity in nucleus magnocellularis of the chicken following cochlea removal.

The metabolism of second order auditory neurons in nucleus magnocellularis (NM) in the chick brainstem can be profoundly altered when excitatory input from the cochlea is removed. Within hours of cochlea removal, NM neurons show an increase in histochemical staining for the Kreb's cycle enzyme succinate dehydrogenase (SDH), followed in several days by decreases in SDH staining. We examined the activity of another Kreb's cycle enzyme, malate dehydrogenase (MDH) using a histochemical stain and a sensitive quantitative biochemical assay for comparison. We found changes in MDH staining similar in time course and magnitude to those of SDH; within 4 h of cochlea removal, MDH activity increases in ipsilateral NM neurons. By 9 days after cochlea removal MDH activity decreases, although not to the same degree as seen with SDH. Biochemical measurements of MDH activity also showed an early increase in activity in ipsilateral NM, followed at 9 days survival by a decrease in activity. Biochemical measurements of the activity of other enzymes in NM may be useful in further defining the metabolic consequences of deafferentation.

Animals↗
PubMed1992

Ontogeny of beta-adrenergic receptors in pulmonary arterial smooth muscle, bronchial smooth muscle, and alveolar lining cells in the rat.

beta-Adrenergic receptors play an integral role in the modulation of cell function in the developing lung. In the rat, there are marked increases in beta receptor density in whole lung during postnatal maturation, but it is now known whether there are differential developmental changes in receptor density in specific cell types. Quantitative light microscopic autoradiography with [125I]iodocyanopindolol ([125I]ICYP) was used to determine maturational changes in beta-adrenergic receptor density in pulmonary arterial smooth muscle (ASM), bronchial smooth muscle (BSM), and alveolar lining cells (ALC) in rat lung during postnatal development (1 day to 6 mo). [125I]ICYP binding to whole lung sections revealed a single class of high-affinity receptors; agonist competitive binding studies suggested that the receptors are primarily of the beta 2 subtype. beta-Adrenergic receptor density in newborn (1 day) lung was lowest in ASM cells and was comparable in BSM cells and ALC. In contrast, in lungs from adult rats (3 mo), receptor density was similar in ASM versus BSM cells and was 2-fold greater in ALC. In addition, the maturational pattern of increasing receptor density differed in ASM compared with BSM and ALC. Receptor density in ASM increased 93% from 1 to 13 days, another 92% from 13 to 20 days, and was unchanged thereafter. In contrast, receptor density in BSM cells did not change from 1 to 13 days, but it increased 65% from 13 to 20 days, rose another 47% from 20 days to 3 mo, and increased an additional 24% from 3 to 6 mo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Afferent influences on brainstem auditory nuclei of the chick: nucleus magnocellularis neuronal activity following cochlea removal.

Elimination of presynaptic elements often results in marked changes, such as atrophy and death, in postsynaptic neurons in the central nervous system. These transneuronal changes are particularly rapid and profound in young animals. In order to understand the cellular events underlying transneuronal regulation it is necessary to explore changes in the local environment of neurons following manipulations of their afferents. In previous investigations we have documented a variety of rapid and marked cellular changes in neurons of the cochlear nucleus of neonatal chicks (n. magnocellularis) following cochlea removal. In adult chickens, however, these transneuronal changes are either absent or minor. The goals of the studies presented here were to examine changes in the electrical activity of nucleus magnocellularis cells and their afferents following removal of the cochlea and to determine if these changes were similar in adult and neonatal animals. Two measures of electrical activity were used; multiunit recording with microelectrodes and incorporation of radiolabeled 2-deoxyglucose (2-DG). Microelectrode recordings revealed high levels of spontaneous activity in n. magnocellularis and n. laminaris, the binaural target of n. magnocellularis neurons. Neither puncturing of the tympanic membrane nor removal of the columella causes significant changes in spontaneous activity, although the latter results in a profound hearing loss (40-50 dB). Removal of the cochlea, on the other hand, results in immediate cessation of all extracellular electrical activity in the ipsilateral n. magnocellularis. Recordings from the same location for up to 6 h failed to reveal any return of spontaneous activity. When the electrode tip was placed in n. laminaris, unilateral cochlea removal had no discernible effect on extracellularly recorded spontaneous activity, probably due to the high levels of excitatory input from the intact ear. Bilateral cochlea removal, however, completely eliminated activity in n. laminaris. 2-DG studies conducted 1 h to 8 days following unilateral cochlea removal revealed marked decreases in 2-DG incorporation in the ipsilateral n. magnocellularis and bilaterally in the n. laminaris target of the ablated cochlea. No compensatory return of 2-DG incorporation was observed for up to 8 days. Comparisons of adult and neonatal chicks failed to reveal significant differences in the effects of cochlea removal on multiunit activity or 2-DG incorporation, suggesting that age differences in transneuronal regulation are due to intrinsic biochemical differences in young and adult neurons rather than differences in the proportion of synaptic input that has been abolished.

Acoustic Stimulation↗
PubMed1990

Effects of cochlea removal on GABAergic terminals in nucleus magnocellularis of the chicken.

The effects of unilateral cochlea removal on GABA-immunoreactive (GABA-I) terminals in nucleus magnocellularis (NM) of the chick were assessed by immunocytochemical (ICC) techniques. Posthatch chicks (5-8 days old) survived from 1-37 days following unilateral cochlea removal. In the ipsilateral NM, the density of GABA-I terminals appeared to increase relative to normal controls 10-37 days after cochlea removal. However, most of that increase could be attributed to a decrease in cell size, cell number, and volume of the nucleus as a result of deafferentation. In the contralateral NM, the density of GABA-I terminals decreased relative to the ipsilateral NM and to normal animals 1-21 days after cochlea removal. The number of GABA-I terminals per NM neuron also decreased in the contralateral NM while that in the ipsilateral NM was comparable to normal controls. To ascertain whether these changes represented changes in the number of terminals or in the amount of GABA contained within the terminals, we also examined these terminals using an antibody to glutamic acid decarboxylase (GAD), the biosynthetic enzyme for GABA. Following unilateral cochlea removal, there was no difference in the density of GAD-I terminals in NM between the two sides of the brain for any of the survival times. Similarly, bilateral cochlea removal had no discernible effect on the density of GABA-I terminals in NM. These data suggest that unilateral deafferentation may temporarily downregulate the biosynthesis of GABA in the contralateral NM.

Animals↗
PubMed1990

Cytochrome oxidase response to cochlea removal in chicken auditory brainstem neurons.

Changes in cytochrome oxidase (CO) activity were studied in the chick brainstem auditory nuclei, n. magnocellularis (NM) and n. laminaris (NL), following unilateral cochlea removal. Chickens aged 10 days or 56 weeks underwent unilateral cochlea removal. Following survival periods of 30 minutes to 14 days for the 10-day-old birds and 6 hours or 14 days for the 56-week-old birds, the animals were perfused with paraformaldehyde/glutaraldehyde fixative. Cryostat sections of the brainstem were then prepared for CO histochemistry. Microdensitometry was used to quantify the difference in CO staining in NM and NL ipsilateral and contralateral to the cochlea removal. Since the cochlea projects to the ipsilateral NM, the contralateral NM was used as a within-animal control. In normal chickens, NM cell bodies and the cell bodies and dendrites of NL neurons stain darkly for CO in both young and adult birds. In 10-day-old birds, there is no significant change in CO staining in NM from 30 minutes to 3 hours after cochlea removal. Then, a rapid biphasic change in CO staining was found in the ipsilateral NM. An increase in staining was observed 6 to 24 hours postoperatively, followed by a decrease in CO staining at 3- to 14-day survival times. In the 56-week-old birds, no increases in CO staining were observed 6 hours after cochlea removal, but a decrease in CO staining was found 14 days postoperatively. In NL, no changes were observed until 3 days (10-day-old birds) or 14 days (56-week-old birds) after cochlea removal. Then a decrease in CO staining was observed in the dendritic and glial/fiber regions of NL containing axons from the deafferented NM. Thus it appears that afferent input has a regulatory effect on the oxidative metabolism of neurons in the chicken auditory brainstem nuclei, an effect that differs with the age of the animal at the time of afferent manipulation.

Age Factors↗