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Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-β to the activin A pathway. We found that TGF-β upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

In the developing nervous system, cell death is an important component of refining axonal projections. In the developing rat inferior olive, previous studies have demonstrated cell death as temporally incongruent with both initial axon-target interactions and subsequent axon collateral regression. Furthermore, these studies identified a late rise in neuron numbers that is concurrent with climbing fibre regression. As axonal regression has not previously been associated with increasing neuron numbers, and since immature neurons and glia have similar morphological characteristics, it was decided to reassess the timing of cell death within the inferior olive in animals in which neurons and glia had been differentially stained. Glia were identified by the presence of glial cytoskeletal proteins, S100, or glial fibrillary acidic protein, and stereological counts were made of both neurons and glia in the inferior olive from rats of ages 0, 5, 10, 15, and 30 days. The number of inferior olivary neurons was approximately 22,000 between birth and day 10, which decreased to about 17,500 by day 30 (P<0.05). In contrast, the number of glia rose from about 5,000 at birth to approximately 15,000 by day 10 (P<0.001), after which there was no further increase. The changes in neurons and glia caused the neuron-to-glia ratio to fall to approximately 1.5 by the time of functional maturation within the olive. These results confirm that there is neuronal death in the inferior olive but that it is temporally correlated with both climbing fibre regression and functional maturation of the olivocerebellar projection.

Alcohol taken regularly over a lengthy period of time has been claimed to cause the loss of neurons in both the adult and developing brain. However, it remains uncertain whether acute, as opposed to chronic, exposure to alcohol at specified periods can also cause disruption in the neuronal population of the developing brain. This question was investigated by exposing Wistar rat pups to 7.5 g/kg body weight of ethanol administered as a 10% solution via an intragastric cannula over an 8 hour period either on the 5th (PND5) or the 10th (PND10) postnatal day of age. Gastrostomy controls received a 5% sucrose solution substituted isocalorically for the ethanol. Another set of pups raised by their mothers was used as "suckle controls." All surgical procedures were carried out under halothane vapour anaesthesia. After the artificial feeding regimes, all pups were returned to the lactating dams and weaned at 21 days of age. Between 52 and 54 days of age, the rats were anaesthetised with an intraperitoneal injection with Nembutal and killed by intracardiac perfusion with 3% glutaraldehyde in 0.1 M phosphate buffer. The relatively unbiased stereological procedure known as the "fractionator" method was used to estimate the total number of Purkinje cells in the cerebellum of each animal. The Purkinje cell nucleolus was used as the counting unit; it was assumed that each Purkinje cell contained only one nucleolus. PND10 ethanol-treated rats and gastrostomy and suckle controls had between about 210,000-232,000 Purkinje cells in the cerebellum. However, the PND5 ethanol-treated rats had only about 137,000 Purkinje cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Undernutrition during early life causes deficits and distortions of brain structure. However, whether or not this includes a diminution of the total numbers of neurones remains uncertain. Recent advances in stereological techniques have made it possible to obtain unbiased estimates of total numbers of cells in well-defined biological structures. Rats were undernourished from conception to 90 postnatal days of age by standardised procedures. Groups of well-fed control and undernourished rats were anaesthetised and killed by intracardiac perfusion with fixatives at 30 and 90 days of age. Each olfactory bulb was serially sectioned at a nominal thickness of 100 microns on a vibratome. These sections were analysed by the Cavalieri principle to obtain estimates of the total volume of the olfactory bulb as well as the volume of its granule cell layer. The physical "disector" method was later used on serial 1-micron-thick toluidine-blue-stained sections to estimate the numerical density of granule cell neurones in the olfactory granule cell layer. These values were used to compute estimates of the total number of olfactory granule cell neurones for each animal. Thirty-day-old control and undernourished rats had between 2.6 and 3 million granule cell neurones in the olfactory bulb. By 90 days of age the number of granule cells had increased in both groups of animals to between about 4.2 and 5.2 million cells. Analysis of variance tests showed a significant main effect of age but not nutrition in these estimates. Although the interaction term did reach statistical significance, post hoc analysis did not reveal any differential effect of undernutrition between the two age groups examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Undernutrition during early life is known to cause deficits and distortions in brain structure. However, it remains uncertain whether this includes a diminution of the total numbers of neurons. Recent advances in stereological techniques have made it possible to obtain unbiased estimates of total numbers of cells in well-defined biological structures. Rats were undernourished from day 16 of gestation to 30 postnatal days of age by standardized procedures. These rats and well-fed control rats were anaesthetized and killed by intracardiac perfusion with fixatives at 70 days of age. The left cerebral hemisphere from each animal was embedded in Paraplast and serially sectioned. The sections were analyzed via the Cavalieri principle to obtain the total cortical volume and by the "disector" method to estimate the numerical density of neurons in the cortex. These values were later used to compute estimates of the total number of cortical neurons for each animal. Well-fed control rats had 26.9 million cortical neurons, while the previously undernourished animals had 24.8 million. The difference between these two groups was not statistically significant. It therefore appears that undernutrition of rats during early postnatal life does not affect the total numbers of neurons in the cerebral cortex.

Undernutrition during early life is known to affect the morphology of the hippocampal formation. Recent advances in stereological techniques have made it possible to make relatively unbiased estimates of total cell numbers in well-defined brain regions. It was decided to use these methods to determine the effects of different levels of undernutrition during early postnatal life on the granule cells of the rat dentate gyrus. Male hooded Long Evans rats were undernourished between the 16th day of gestation and 30 postnatal days of age to two different levels. The daily food intake of level-1 and level-2 rats represented about 60 and 40%, respectively, of that eaten by well-fed, age-matched controls. Nutritional rehabilitation of the rats was commenced when they had reached 30 days of age by placing them on an ad libitum diet. Groups of control and experimental rats were killed at 70 and 212 days of age. The Cavalieri principle was used to determine the granule cell layer volume within the dentate gyrus, and the "dissector" method was used to determine numerical densities of these granule cells. These estimates were used to calculate the total numbers of granule cells. There were between 260,000 and 320,000 granule cells within the dentate gyrus of 70-day-old control and experimental rats. By 212 days of age, well-fed controls had an average of about 834,000 granule cells. The level-1 and level-2 previously undernourished rats had about 515,000 and 595,000 granule cells, respectively. Two-way analysis of variance procedures showed significant main effects of nutrition and age as well as a significant interaction between them.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats were undernourished from the 16th day of gestation until 25 postnatal days of age and then weaned on to an ad libitum diet. Around 35 days of age, 12 previously undernourished male rats were assigned to an enriched environmental condition (EC) and their littermates to an isolated environmental condition (IC). A parallel set of well-fed rats was similarly assigned. After 80 days in these environmental conditions, all rats were killed by perfusion with 2% phosphate buffered glutaraldehyde. Small pieces of tissue containing the entire depth of the right visual cortex were embedded in Spurr's resin. Semithin (0.5 micron) sections were cut from these blocks and stained with toluidine blue. Photomicrographs of these sections were used to estimate the numerical density of neurons in cortical layers II to III. Ultrathin sections (approximately 70 nm) of the same region of cortex were then prepared for electron microscopy. These were used to estimate the mean synaptic disc diameter and synaptic numerical density. From these estimates of neuronal and synaptic numerical density, synapse-to-neuron ratios were calculated. The results of a two-way analysis of variance test revealed that environment had significant effects on neuronal numerical density, mean synaptic disc diameter, and synapse-to-neuron ratios. Neither nutrition nor its interaction with environment had significant effects on any of the parameters analyzed. These results suggest that environmental diversity can produce alterations in certain neuronal and synaptic characteristics in the visual cortex of both well-fed and previously undernourished rats.

Male rats undernourished from the 18th day of gestation until 100 days of age were nutritionally rehabilitated until 200 days of age. Six control and six experimental rats at each of 12, 25, 50, 100, and 200 days of age were killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of visual cortex from each rat were postfixed in osmium tetroxide and embedded in resin. Stereological procedures at the light and electron microscope levels were used to estimate the synapse-to-neuron ratios in cortical layers II to IV. There were no statistically significant differences in the synapse-to-neuron ratio between control and undernourished rats at 12, 25, and 50 days of age. However 100-day-old undernourished rats had a significant deficit in this ratio compared to age-matched controls. Despite this, 200-day-old nutritionally rehabilitated rats were found to have, on average, 23% more synapses per neuron than controls. In both the control and the undernourished groups the synapse-to-neuron ratio increased to a peak by 50 days of age. This was followed by a significant fall in the ratio by 100 days of age. Although there was no further change in the control rats, the experimental group showed a substantial increase in the ratio by 200 days of age. This latter increase appeared to be related to the period of nutritional rehabilitation.

Male rats undernourished from birth to 30 days of age were nutritionally rehabilitated till 160 days of age. Quantitative stereological procedures at the light microscope level were used to estimate, among other things, the numerical densities of cerebellar granule and Purkinje cells on a "per unit volume of cortex" basis. These were subsequently used to calculate granule-to-Purkinje cell ratios. The 30-day-old undernourished rats had a mean +/- S.E. of 290 +/- 27 granule cells for every Purkinje cell present, compared to 395 +/- 34 for the controls. This was a deficit of about 27% (p < 0.05). At 160 days of age, the previously undernourished rats still showed a persisting deficit of about 25% (p < 0.05) in this ratio, despite the lengthy nutritional rehabilitation. There were no statistically significant age-related changes in this ratio. The numerical density of Purkinje cells, but not that of granule cells, was significantly greater in the previously undernourished rats than in controls, for both age groups, Increasing age caused a fall in the numerical density of both cell types. Granule and Purkinje cell nuclear diameters were unaffected by nutrition. However, Purkinje cell nuclei decreased in size by between 7%--13% with increasing age. These results indicate that undernutrition during early life can cause a permanent distortion of the relative number of the various cell types in the cerebellum.

Groups of pigmented (Black and White Hooded Lister) and albino (Sprague Dawley) rats were killed at 7, 15, and 25 postnatal days of age. Their optic nerves were embedded in resin suitable for both light and electron microscopy. Quantitative stereological procedures were used to estimate total fibre number and the degree of myelination in the optic nerves at the various ages. At 7 days of age, both albino and pigmented rats had about 220,000 optic nerve fibres. By 15 days, both strains showed a reduction of some 30,000 fibres. This fibre loss continued in both strains after 15 days of age, but more rapidly in the pigmented strain. At 25 days of age, pigmented rats had 72,371 +/- 7,244 fibres, whilst albino rats had 102,681 +/- 4,138 fibres (P less than .01). More than 99.5% of optic nerve axons in both strains were unmyelinated at 7 days of age. By 25 days of age about 90% of all remaining fibres were myelinated in both strains. The mean diameter of the myelinated axons was estimated to be between 0.59 and 0.65 micron in all animals, there being no significant age or strain differences. In contrast, the mean diameter of nonmyelinated axons increased significantly with age. This increase was greater for albino than pigmented rats, such that by 25 days of age the respective values were 0.49 micron and 0.42 micron (P less than .05).

Male rats undernourished from birth to 30 days were nutritionally rehabilitated till 160 days of age. Quantitative stereological procedures at the light and electron microscopical levels were employed to estimate, among other things, the synapse-to-neuron ratios in the frontal cortex and granular layer of the cerebellum. In the frontal cortex, the 30-day-old undernourished rat had a mean +/- SE of about 14,020 +/- 1,540 synapses-per-neuron compared with 22,270 +/- 3,250 for the controls. This was a deficit of 37% (p < 0.05). By 160 days of age the previously undernourished rats showed no statistically significant deficit in this ratio compared with controls (11,800 +/- 690 and 13,360 +/- 1,110 respectively, p > 0.1). This was due mainly to a fall in the synapse-to-neuron ratio with age. A much larger fall in the ratio occurred in the control than in the previously undernourished group. In the granular layer of the cerebellum the 30-day-old undernourished rats had 341 +/- 17 synapses-per-neuron compared with 495 +/- 25 for the controls. This was a deficit of 31% (p < 0.01). By 160 days of age the previously undernourished rats again showed no statistically significant deficit in this ratio compared with controls (627 +/- 56 and 688 +/- 38, respectively (p > 0.1). These results show that the previously undernourished rats are capable of at least some (if not complete) "catch-up" with regard to the synapse-to-neuron ratio.

Recent evidence showing alterations in spatial memory due to a period of undernutrition during early life has implicated the hippocampus as one of the brain centres that may be particularly adversely affected. However, there are very few quantitative morphological studies that have examined the neuronal and synaptic populations of the hippocampi from undernourished animals. We decided to carry out such experiments, paying particular attention to the granule cell of the dentate gyrus. Male rats were undernourished from the 18th day of gestation until 21, 75, or 150 days of age. Some of these previously undernourished rats were nutritionally rehabilitated between 150 and 250 days of age. Groups of well-fed control and experimental rats were killed by intracardiac perfusion with 2.5% sodium-cacodylate-buffered glutaraldehyde. The right hippocampus from each rat was dissected out and processed for electron microscopy. Stereological procedures at the light and electron microscopical levels were used to estimate the numerical densities of granular cell neurons and molecular layer synapses in the dorsal lip of the dentate gyrus. These estimates were used to calculate synapse: neuron ratios. There were 5,056 +/- 347 (mean +/- SE) and 5,002 +/- 190 synapses per neuron in 21-day-old control and undernourished rats, respectively. By 75 days these values had increased to 9,215 +/- 588 and 6,683 +/- 237. The difference was statistically significant. By 150 days of age the value for control animals had fallen once again to 6,518 +/- 209 whereas undernourished rats had increased slightly to 7,689 +/- 288 (P less than .01); 250-day-old rats, previously undernourished from birth to 150 days of age, showed a substantial increase in the synapse: neuron ratio during the period of nutritional rehabilitation. Thus these nutritionally rehabilitated rats had 9,407 +/- 365 synapses per neuron whereas age-matched controls had only 6,323 +/- 239 (P less than .01). These results indicate that the rat dentate gyrus is vulnerable to undernutrition even during the postweaning period and that a lengthy period of undernutrition can alter the developmental growth curve for synapse: neuron ratios.

Male rats undernourished from the 16th day of gestation until 25 postnatal days of age were raised either in enriched (EC) or isolated (IC) environmental conditions between about 35 and 115 days of age. A parallel set of well-fed rats was raised in identical environments. At the end of this period all rats were killed by perfusion with 2% phosphate buffered glutaraldehyde. Body and forebrain weights and forebrain lengths and widths were determined for each animal. The left cerebral hemisphere was embedded in paraffin-wax and serially sectioned. Three of these coronal sections (defined by subcortical landmarks) taken from the occipital cortex region were used for cortical depth measurements. Small pieces of cortical tissue taken from area 17 of the right cerebral hemisphere were embedded in resin and cut to yield 0.5 micrometer thick sections through the entire depth of the cortex. These sections were used to estimate the nuclear diameters and numerical densities of neurons and oligodendrocytes as well as neuronal perikaryal volumes. Two-way analysis of variance tests on data combined from both nutritional groups revealed significant main effects of nutrition on body weight, forebrain weight, forebrain width, and forebrain length X width values. Environment had significant main effects on forebrain weight, forebrain length, forebrain length X width values, and on neuronal numerical density. The interaction between nutrition and environment was not significant for any of the measurements carried out. These results suggest that 80 days of environmental diversity beginning at about 35 days of age can produce morphological changes in the brains of both well-fed rats and rats undernourished during early life.(ABSTRACT TRUNCATED AT 250 WORDS)

Black and white Lister hooded rats were undernourished from the 16th day of gestation until 25 postnatal days of age. These previously undernourished rats and a set of well-fed rats were later subjected to 30 days of environmental diversity, i.e., environmental enrichment or isolation. Two separate experiments were carried out. In experiment 1, the environmental diversity lasted from 85 to 115 days of age and in experiment 2, from 35 to 65 days of age. At the end of the period of environmental diversity, all rats were killed by perfusion with 2% phosphate-buffered glutaraldehyde. Small pieces of tissue from the right visual cortex were embedded in Spurr's resin. For each rat two blocks of resin-embedded tissue were randomly selected, and from these semithin sections (0.5 micron) were cut and stained with toluidine blue. Photomicrographs of cortical layers II and III were taken from these sections and used to estimate the numerical density of neurons. Ultrathin (ca. 70 nm) sections of the same region of the cortex were cut and stained with lead citrate. These sections were used to estimate the synaptic disc diameter and numerical density. Synapse-to-neuron ratios were calculated from the estimates of synaptic and neuronal numerical densities. In experiment 1, well-fed rats raised in enriched environments had a significantly smaller neuronal numerical density and a greater synaptic disc diameter than well-fed rats raised in an impoverished environment. In experiment 2, neither the well-fed nor previously undernourished rats showed significant effects of environmental treatment on any of the features studied. The statistical interaction between nutrition and environment was not significant for any of the features in either experiment.

Male rats undernourished from the 18th day of gestation until 100 days of age were nutritionally rehabilitated until 200 days of age. Six control and six experimental rats at each of 100 and 200 days of age were killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of visual cortex from each rat were postfixed in osmium tetroxide and embedded in resin. Stereological procedures at the light and electron microscopy levels were used to estimate the synapse-to-neuron ratios in cortical layers II to IV. Rats undernourished until 100 days of age had a mean +/- S.E. of 10,350 +/- 470 synapses associated with each neuron. This represented a 13% deficit (P less than 0.05) when compared to the control value of 11,950 +/- 530. Following nutritional rehabilitation till 200 days of age it was found that the previously undernourished rats had about 23% more (P less than 0.05) synapses-per-neuron than their age-matched controls. This was due almost entirely to a substantial increase in the ratio in the previously undernourished animals; the value of controls did not alter significantly between the two age groups. It appears that the deficit in the synapse-to-neuron ratio seen after a lengthy period of undernutrition is not permanent, at least in rats subsequently allowed nutritional rehabilitation. In fact, such animals seem to be capable of not only "catching-up" but "overshooting" the values found in age-matched controls.

Male rats undernourished from the 18th day of gestation till 100 days of age were nutritionally rehabilitated until 200 days of age. Six control and six experimental rats at each of 25, 50, 100, and 200 days of age were killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of optic nerve from just behind the left eye of each rat were postfixed in osmium tetroxide and embedded in resin. Estimates of the total number of fibres and their mean diameter and size-frequency distributions were made for each nerve. Rats undernourished until 25 days of age had a mean +/- SE of 57,464 +/- 6,778 fibres per optic nerve. This represented a 36% deficit (P less than 0.01) compared with the 25-day-old control value of 89,778 +/- 6,625. In addition, these undernourished rats had proportionately more small fibres than the age-matched controls. This resulted in a significant deficit in mean fibre diameter at this age. These deficits and distortions disappeared in all the older rats studied, despite the continued undernourishment of some animals up to 100 days of age. It appears that even the lengthy period of undernutrition imposed in the present experiments could not produce a permanent deficit in optic nerve fibre number, size, and size distribution. It is suggested that the deficits seen initially at 25 days of age may have been due to a temporary delay in the growth and development of these nerve fibres.

Rats whose right eyes were enucleated on day 1 after birth and nonenucleated rats were raised in either "light" or "dark" (red light) conditions from birth until 39 days of age. This resulted in 4 groups of animals: light-reared nonenucleated, light-reared enucleated, dark-reared nonenucleated, and dark-reared enucleated. At 39 days of age, the animals were killed by perfusion with 2.5% sodium cacodylate buffered glutaraldehyde. The superior colliculi were dissected out and processed for embedding in resin. Stereological procedures at the light and electron microscopical levels were used to estimate the synapse-to-neuron ratios in the superficial layers of these colliculi. Light-reared, nonenucleated rats had about 1,850 synapses-per-neuron in both the right and left superior colliculi. Rearing nonenucleated rats in the dark reduced this value to about 1,200. Enucleated rats reared in the light showed a differential response in the 2 colliculi. Thus, the contralateral (to the enucleated eye) colliculi showed a decrease, whereas the ipsilateral colliculi showed an increase in the synapse-to-neuron ratio compared with light-reared, nonenucleated rats. When enucleated rats were reared in the red light, there was a decrease in the ratio in both colliculi, although the extent of this decrease was more marked in the contralateral than the ipsilateral colliculi. However, the decrease in the contralateral colliculi was not significantly greater than that observed in the corresponding colliculi from dark-reared, nonenucleated rats. These results provide useful information on the combined and separate effects of unilateral enucleation at around birth and dark (red light) rearing during early life on the interneuronal connectivity of both the ipsi- and contralateral superior colliculi of rats. They also show the vast importance of visual stimulation for the normal development of the subcortical visual centers.

Stereological estimates of the numbers of Purkinje cell nucleoli in human cerebellar cortex have been obtained from systematic random samples of tissue by using the fractionator. The estimates are unbiased by fixation, section thickness, or sampling errors and are independent of any assumptions about cell shape, size, or spatial orientation. Twelve brains from aged subjects of both sexes were examined. The average complement of nucleoli in four female brains (age range 71-93 years) amounted to 14.8 millions (with an observed coefficient of variation between subjects of 29%). For three male brains (76-91 years), the corresponding estimates were 15.7 millions (10%). No significant sex differences were found for these small samples. Five brains of unknown sex and age yielded values of 15.8 millions (18%). For the twelve brains examined, the total number of Purkinje cell nucleoli per cerebellum was found to be 15.4 millions (19%). Estimated numbers showed a significant positive correlation with cerebellar weights. The number of nucleoli in an individual cerebellum was obtained with high precision in as short a time as 4 hours.

It is possible that the reported effects of early life undernutrition on brain morphology may be due to alterations in mother-infant interactions and not directly to undernutrition. We have investigated this possibility by comparing artificially reared with mother-reared rats. Four groups of black-and-white hooded male rats were reared. These consisted of mother reared control (MRC), mother reared undernourished (MRU), artificially reared control (ARC) and artificially reared undernourished (ARU). Artificially reared rats were raised in isolation away from their mothers from 5 to 21 days of postnatal age. They were fitted with a gastric cannula through which 'milk' was infused automatically. The period of undernutrition lasted from 5 to 25 postnatal days, following which the animals were fed ad libitum until 312 days of age. Rats from each group were then killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of visual cortex from each rat were postfixed in osmium tetroxide and embedded in resin. Stereological procedures at the light and electron microscopical levels were used to estimate the synapse-to-neuron ratios in cortical layers II to IV. Both MRC and ARC rats had about 7000 synapses per neuron. However, this ratio was about 8300 in MRU rats whilst it was only about 5000 in ARU animals. The rearing x nutrition interaction was statistically significant at the 0.1% level. These changes in the synapse-to-neuron ratio were mainly due to alterations in the numerical densities of the synapses rather than that of neurons. These results demonstrate that environmental isolation, as a result of artificial rearing procedures, and concurrent undernutrition during the first three weeks of postnatal life, interact with one another to produce marked morphological changes in the adult rat brain. However, environmental isolation was not, by itself, sufficient to cause permanent changes in interneuronal connectivity.