Evaluation of PET imaging as a tool for detecting neonatal hypoxic-ischemic encephalopathy in a preclinical animal model.

Hypoxic-ischemic encephalopathy due to insufficient oxygen delivery to brain tissue is a leading cause of death or severe morbidity in neonates. The early recognition of the most severely affected individuals remains a clinical challenge. We hypothesized that hypoxic-ischemic injury can be detected using PET radiotracers for hypoxia ([18F]EF5), glucose metabolism ([18F]FDG), and inflammation ([18F]F-DPA). METHODS: A preclinical model of neonatal hypoxic-ischemic brain injury was made in 9-d-old rat pups by permanent ligation of the left common carotid artery followed by hypoxia (8% oxygen and 92% nitrogen) for 120 min. In vivo PET imaging was performed immediately after injury induction or at different timepoints up to 21 d later. After imaging, ex vivo brain autoradiography was performed. Brain sections were stained with cresyl violet to evaluate the extent of the brain injury and to correlate it with [18F]FDG uptake. RESULTS: PET imaging revealed that all three of the radiotracers tested had significant uptake in the injured brain hemisphere. Ex vivo autoradiography revealed high [18F]EF5 uptake in the hypoxic hemisphere immediately after the injury (P < 0.0001), decreasing to baseline even 1 d postinjury. [18F]FDG uptake was highest in the injured hemisphere on the day of injury (P < 0.0001), whereas [18F]F-DPA uptake was evident after 4 d (P = 0.029), peaking 7 d postinjury (P < 0.0001), and remained significant 21 d after the injury. Targeted evaluation demonstrated that [18F]FDG uptake measured by in vivo imaging 1 d postinjury correlated positively with the brain volume loss detected 21 d later (r = 0.72, P = 0.028). CONCLUSION: Neonatal hypoxic-ischemic brain injury can be detected using PET imaging. Different types of radiotracers illustrate distinct phases of hypoxic brain damage. PET may be a new useful technique, worthy of being explored for clinical use, to predict and evaluate the course of the injury.

Small animal PET with spontaneous inhalation of 15O-labelled oxygen gases: Longitudinal assessment of cerebral oxygen metabolism in a rat model of neonatal hypoxic-ischaemic encephalopathy.

Perinatal hypoxic-ischaemic encephalopathy (HIE) is the leading cause of irreversible brain damage resulting in serious neurological dysfunction among neonates. We evaluated the feasibility of positron emission tomography (PET) methodology with 15O-labelled gases without intravenous or tracheal cannulation for assessing temporal changes in cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) in a neonatal HIE rat model. Sequential PET scans with spontaneous inhalation of 15O-gases mixed with isoflurane were performed over 14 days after the hypoxic-ischaemic insult in HIE pups and age-matched controls. CBF and CMRO2 in the injured hemispheres of HIE pups remarkably decreased 2 days after the insult, gradually recovering over 14 days in line with their increase found in healthy controls according to their natural maturation process. The magnitude of hemispheric tissue loss histologically measured after the last PET scan was significantly correlated with the decreases in CBF and CMRO2.This fully non-invasive imaging strategy may be useful for monitoring damage progression in neonatal HIE and for evaluating potential therapeutic outcomes.

Overlap between EEC and AEC syndrome and immunodeficiency in a preterm infant with a TP63 variant.

Pathogenic variants in the transcription factor TP63 gene cause a variety of clinical phenotypes, such as ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome and ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome. Historically, TP63-related phenotypes have been divided into several syndromes based on both the clinical presentation and location of the pathogenic variant on the TP63 gene. This division is complicated by significant overlap between syndromes. Here we describe a patient with clinical characteristics of different TP63-associated syndromes (cleft lip and palate, split feet, ectropion, erosions of the skin and corneas), associated with a de novo heterozygous pathogenic variant c.1681 T>C, p.(Cys561Arg) in exon 13 of the TP63 gene. Our patient also developed enlargement of the left-sided cardiac compartments and secondary mitral insufficiency, which is a novel finding, and immune deficiency, which has only rarely been reported. The clinical course was further complicated by prematurity and very low birth weight. We illustrate the overlapping features of EEC and AEC syndrome and multidisciplinary care needed to address the various clinical challenges.

NIV-NAVA versus NCPAP immediately after birth in premature infants: A randomized controlled trial.

OBJECTIVE: To evaluate whether noninvasive-neurally adjusted ventilatory assist (NIV-NAVA) decrease respiratory efforts compared to nasal continuous positive airway pressure (NCPAP) during the first hours of life. METHODS: Twenty infants born between 28+0 and 31+6 weeks were randomized to NIV-NAVA or NCPAP. Positive end-expiratory pressure was constantly kept at 6 cmH2O for both groups and the NAVA level was 1.0 cmH2O/µV for NIV-NAVA group. The electrical activity of diaphragm (Edi) were recorded for the first two hours. RESULTS: Peak and minimum Edi decreased similarly in both groups (P = 0.98 and P = 0.59, respectively). Leakages were higher in the NIV-NAVA group than in the NCPAP group (P < 0.001). The neural apnea defined as a flat Edi for ≥ 5 s were less frequent in NIV-NAVA group than in NCPAP group (P = 0.046). CONCLUSIONS: Immediately applied NIV-NAVA in premature infants did not reduce breathing effort, measured as peak Edi. However, NIV-NAVA decreased neural apneic episodes compared to NCPAP.

Implementation of neurally adjusted ventilatory assist and high flow nasal cannula in very preterm infants in a tertiary level NICU.

Preterm infants treated with invasive ventilation are often affected by bronchopulmonary dysplasia, brain structure alterations, and later neurodevelopmental impairment. We studied the implementation of neurally adjusted ventilatory assist (NAVA) and high flow nasal cannula (HFNC) in a level III neonatal unit, and its effects on pulmonary and central nervous system outcomes. This retrospective cohort study included 193 surviving infants born below 32 weeks of gestation in preimplementation (2007-2008) and postimplementation (2016-2017) periods in a single study center in Finland. The proportion of infants requiring invasive ventilation decreased from 67% in the pre- to 48% in the postimplementation period (p = 0.009). Among infants treated with invasive ventilation, 68% were treated with NAVA after its implementation. At the same time, the duration of invasive ventilation of infants born at or below 28 weeks increased threefold compared with the preimplementation period (p = 0.042). The postimplementation period was characterized by a gradual replacement of nasal continuous positive airway pressure (nCPAP) with HFNC, earlier discontinuation of nCPAP, but a longer duration of positive pressure support. The proportion of normal magnetic resonance imaging (MRI) findings at term corrected age increased from 62% to 84% (p = 0.018). Cognitive outcome improved by one standard score between the study periods (p = 0.019). NAVA was used as the primary mode of ventilation in the postimplementation period. During this period, invasive ventilation time was significantly prolonged. HFNC led to a decrease in the use of nCPAP. The change in the respiratory support might have contributed to the improvement in brain MRI findings and cognitive outcomes.

Parent-infant skin-to-skin contact reduces the electrical activity of the diaphragm and stabilizes respiratory function in preterm infants.

BACKGROUND: The physiological benefit of parent-infant skin-to-skin contact (SSC) is uncertain for preterm infants with ventilatory support. We aimed to investigate whether SSC stabilizes the respiration compared to incubator care in mechanically ventilated preterm infants. METHODS: The prospective observational study was performed in Turku University Hospital, Finland. Preterm infants were eligible if they were born before 36 weeks gestation and received respiratory support with either invasive or non-invasive neurally adjusted ventilatory assist (NAVA). SSC was applied as soon as possible after birth. Respiratory variables were collected from the ventilator log data, and SSC episodes were compared with matched control periods during incubator care. RESULTS: A total of 167 episodes of SSC were recorded from 17 preterm infants: 138 episodes during invasive NAVA and 29 episodes during non-invasive NAVA. During invasive NAVA, peak electrical activity of the diaphragm (Edi), minimum Edi, respiratory rate, time on backup ventilation, peak inspiratory pressure, and mean airway pressure were significantly lower in SSC than in incubator care. During non-invasive NAVA, peak Edi, minimum Edi, time on backup ventilation, and peak inspiratory pressure were significantly lower in SSC than in incubator care. CONCLUSIONS: SSC stabilized and improved the respiratory physiology in mechanically ventilated preterm infants. IMPACT: Skin-to-skin contact reduced work of breathing compared to incubator care in mechanically ventilated preterm infants. Skin-to-skin contact reduced the need for backup ventilation during neurally adjusted ventilatory assist in preterm infants. Skin-to-skin contact among ventilated preterm infants was not only safe but also stabilized and improved their respiratory physiology.

Neurally adjusted ventilatory assist in ventilated very preterm infants: A crossover study.

OBJECTIVE: To assess the effects of neurally adjusted ventilatory assist (NAVA) ventilation on oxygenation and respiratory parameters in preterm infants. STUDY DESIGN: An observational crossover study with a convenience sample of 19 infants born before 30 gestational weeks. Study parameters were recorded during 3-h periods of both NAVA and conventional ventilation. The proportion of time peripheral oxygen saturation (SpO2 ) and cerebral regional oxygen saturation (cRSO2 ) were within their target ranges, plus the number and severity of desaturation episodes were analyzed. In addition, electrical activity of the diaphragm (Edi), neural respiratory rates, and peak inspiratory pressures (PIPs) were recorded. RESULTS: Infants were born at a median age of 264/7 gestational weeks (range: 230/7 -293/7 ); the study was performed at a median age of 20 days (range: 1-82). The proportion of time SpO2 was within the target range, the number of peripheral desaturations or cRSO2 did not differ between the modes. However, the desaturation severity index was lower (131 vs. 152; p = .03) and fewer manual supplemental oxygen adjustments (1.3 vs. 2.2/h; p = .006) were needed during the period of NAVA ventilation following conventional ventilation. The mean Edi (8.1 vs. 11.4 µV; p < .006) and PIP values (14.9 vs. 19.1; p < .001) were lower during the NAVA mode. CONCLUSIONS: Although NAVA ventilation did not increase the proportion of time with optimal saturation, it was associated with decreased diaphragmatic activity, lower PIPs, less severe hypoxemic events, and fewer manual oxygen adjustments in very preterm infants.

Backup ventilation during neurally adjusted ventilatory assist in preterm infants.

OBJECTIVE: To analyze the proportion of backup ventilation during neurally adjusted ventilatory assist (NAVA) in preterm infants at different postmenstrual ages (PMAs) and to analyze the trends in backup ventilation in relation to clinical deteriorations. METHODS: A prospective observational study was conducted in 18 preterm infants born at a median (range) 27+4 (23+4 -34+4 ) weeks of gestation with a median (range) birth weight of 1,100 (460-2,820) g, who received respiratory support with either invasive or noninvasive NAVA. Data on ventilator settings and respiratory variables were collected daily; the mean values of each 24-h recording were computed for each respiratory variable. For clinical deterioration, ventilator data were reviewed at 6-h intervals for 30 h before the event. RESULTS: A total of 354 patient days were included: 269 and 85 days during invasive and noninvasive NAVA, respectively. The time on backup ventilation (%/min) significantly decreased with increasing PMA during both invasive and noninvasive NAVA. The neural respiratory rate did not change over time. The median time on backup ventilation was less than 15%/min, and the median neural respiratory rate was more than 45 breaths/min for infants above 26+0 weeks PMA during invasive NAVA. The relative backup ventilation significantly increased before the episode of clinical deterioration. CONCLUSION: The proportion of backup ventilation during NAVA showed how the control of breathing matured with increasing PMA. Even the most immature infants triggered most of their breaths by their own respiratory effort. An acute increase in the proportion of backup ventilation anticipated clinical deterioration.

Nasal high-flow therapy decreased electrical activity of the diaphragm in preterm infants during the weaning phase.

AIM: We evaluated whether nasal high-flow therapy was better than no respiratory support during the weaning phase in preterm infants. METHODS: The study was conducted in the neonatal intensive care unit of the Turku University Hospital between September 2014 and August 2015. Preterm infants who were alternating between nasal high-flow therapy and unassisted breathing were enrolled. Electrical activity of the diaphragm (EAdi) was recorded and compared during three-hour time periods for each option. RESULTS: We studied eight infants at a median gestational age of 31 weeks. The EAdi peak was lower during nasal high-flow therapy when compared to no respiratory support (6.1 µV vs 7.1 µV, p = 0.02), but the EAdi minimum was similar with and without respiratory support. Neural respiratory rate (62 vs 68 per minute, p = 0.02) and the frequency of sighs (27.8 vs 37.9 per hour, p = 0.03) were lower during nasal high-flow therapy than no respiratory support. CONCLUSION: Nasal high-flow therapy reduced diaphragm activation in our cohort when compared to no respiratory support, as indicated by the lower Edi peak. An increase in the respiratory rate and the sigh frequency without respiratory support also suggests that nasal high-flow therapy provided support during the weaning phase.

Rapid respiratory transition at birth as evaluated by electrical activity of the diaphragm in very preterm infants supported by nasal CPAP.

OBJECTIVE: To investigate breathing patterns during respiratory adaptation in preterm infants using the electrical activity of the diaphragm (EAdi) signal. PATIENTS: Infants born between 28 + 0 and 31 + 6 gestational weeks and supported by early nasal continuous positive airway pressure (nCPAP) were studied. The EAdi signal was recorded for 120 min after birth. RESULTS: Eight preterm infants were evaluated. The median EAdi peak value of 19.2 µV (lower quartile 13.1; upper quartile 22.2) at 20 min after birth decreased to 11.4 µV (9.5-14.7) at 55 min of age. The median EAdi minimum value of 4.5 µV (2.2-5.5) at 25 min after birth decreased to 1.6 µV (1.2-2.7) at 85 min of age. CONCLUSION: EAdi was high right after birth. This indicates that preterm infants are capable of generating sufficient respiratory drive and diaphragm tone during expiration to establish and maintain functional residual capacity. Diaphragm activity decreased within the first 90 min, suggesting that early adaptation was accomplished by 90 min of age.

The effect of caffeine citrate on neural breathing pattern in preterm infants.

BACKGROUND: Caffeine citrate is widely used to prevent and treat prematurity-associated apnea. AIMS: The aim of this study was to characterize the effect of caffeine citrate on the neural control of breathing, especially central apnea, in premature infants. STUDY DESIGN: Preterm infants were evaluated for 30min before and 30min after caffeine citrate loading (20mg/kg). A feeding tube including miniaturized sensors was used to measure the diaphragm electrical activity (Edi) waveform. Central apnea was defined as any period where the Edi waveform was flat for >5s. SUBJECTS: Seventeen preterm infants with a mean age of three days and mean birth weight of 900 grams were evaluated. OUTCOME MEASURES: In addition to central apnea, several parameters including neural inspiratory time, neural respiratory rate, peak Edi, delta inspiratory change in Edi (phasic Edi) and minimum Edi on exhalation were measured. RESULTS: The majority of the apnea were short (5 to 10s) and the number of apnea correlated with birth weight (p=0.039). Caffeine citrate reduced significantly the number of 5-to-10-second-long central apnea during the 30-minute periods (12±11 to 7±7; p=0.02). Caffeine citrate increased both peak and phasic Edi leading to a significant increase in the diaphragm energy expenditure. CONCLUSIONS: Edi signal can be reliably measured and processed to study changes in premature infants' neural breathing. The beneficial effect of caffeine citrate on the reduction of the number of apnea is mediated through stimulated neural breathing increasing the diaphragm energy expenditure.

Systemic T-helper and T-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study.

BACKGROUND: Rhinovirus (RV) associated early wheezing has been recognized as an independent risk factor for asthma. The risk is more important than that associated with respiratory syncytial virus (RSV) disease. No comparative data are available on the immune responses of these diseases. OBJECTIVE: To compare T-helper1 (Th1), Th2 and T-regulatory (Treg) cell type cytokine responses between RV and RSV induced early wheezing. METHODS: Systemic Th1-type (interferon [IFN] -gamma, interleukin [IL] -2, IL-12), Th2-type (IL-4, IL-5, IL-13) and Treg-type (IL-10) cytokine responses were studied from acute and convalescence phase serum samples of sole RV (n = 23) and RSV affected hospitalized wheezing children (n = 27). The pre-defined inclusion criteria were age of 3-35 months and first or second wheezing episode. Analysis was adjusted for baseline differences. Asymptomatic children with comparable demographics (n = 11) served as controls for RV-group. RESULTS: RV-group was older and had more atopic characteristics than RSV-group. At acute phase, RV-group had higher (fold change) IL-13 (39-fold), IL-12 (7.5-fold), IFN-gamma (6.0-fold) and IL-5 (2.8-fold) concentrations than RSV-group and higher IFN-gamma (27-fold), IL-2 (8.9-fold), IL-5 (5.6-fold) and IL-10 (2.6-fold) than the controls. 2-3 weeks later, RV-group had higher IFN-gamma (>100-fold), IL-13 (33-fold) and IL-10 (6.5-fold) concentrations than RSV-group and higher IFN-gamma (15-fold) and IL-2 (9.4-fold) than the controls. IL-10 levels were higher in acute phase compared to convalescence phase in both infections (p < 0.05 for all). CONCLUSION: Our results support a hypothesis that RV is likely to trigger wheezing mainly in children with a predisposition. IL-10 may have important regulatory function in acute viral wheeze.

Dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children who later progress to type 1 diabetes.

The risk determinants of type 1 diabetes, initiators of autoimmune response, mechanisms regulating progress toward beta cell failure, and factors determining time of presentation of clinical diabetes are poorly understood. We investigated changes in the serum metabolome prospectively in children who later progressed to type 1 diabetes. Serum metabolite profiles were compared between sample series drawn from 56 children who progressed to type 1 diabetes and 73 controls who remained nondiabetic and permanently autoantibody negative. Individuals who developed diabetes had reduced serum levels of succinic acid and phosphatidylcholine (PC) at birth, reduced levels of triglycerides and antioxidant ether phospholipids throughout the follow up, and increased levels of proinflammatory lysoPCs several months before seroconversion to autoantibody positivity. The lipid changes were not attributable to HLA-associated genetic risk. The appearance of insulin and glutamic acid decarboxylase autoantibodies was preceded by diminished ketoleucine and elevated glutamic acid. The metabolic profile was partially normalized after the seroconversion. Autoimmunity may thus be a relatively late response to the early metabolic disturbances. Recognition of these preautoimmune alterations may aid in studies of disease pathogenesis and may open a time window for novel type 1 diabetes prevention strategies.

Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX) and are widely used for post-trauma musculoskeletal analgesia. In animal models, NSAIDs have been reported to delay fracture healing and cause non-union, possibly due to the drug-induced inhibition of osteoblast recruitment and differentiation. To further investigate the cellular effects of these drugs in the context of bone healing, we examined the effects of COX-1 inhibitor indomethacin and COX-2 inhibitors, parecoxib and NS398 on osteoclast and osteoblast differentiation and activity in vitro. We discovered that all tested COX-inhibitors significantly inhibited osteoclast differentiation, by 93%, 94% and 74% of control for 100 microM indomethacin, 100 microM parecoxib and 3 microM NS398, respectively. Furthermore, inhibition of COX-2 reduced also the resorption activity of mature osteoclasts. All tested COX-inhibitors also significantly inhibited osteoblast differentiation from human mesenchymal stem cells. Simultaneously, the number of adipocytes was significantly increased. The adipocyte covered areas in the cultures with 1 microM indomethacin, 1 microM parecoxib and 3 microM NS398 were 9%, 29% and 24%, respectively, as compared with 6% in the control group. This data suggests that COX-2 inhibition disturbs bone remodelling by inhibiting osteoclast differentiation and diverting stem cell differentiation towards adipocyte lineage instead of osteoblast lineage. In conclusion, our results further suggest cautious use of COX-2 inhibitors after osseous trauma.

Intact surface of bioactive glass S53P4 is resistant to osteoclastic activity.

Bioactive glass reacts with body fluids and is gradually dissolved in tissues and in cell cultures. We investigated whether osteoclasts contribute to this process, by culturing newborn rat bone-marrow cells containing osteoclasts on polished bioactive glass plates (glass S53P4). The cultures were inspected at days 1-5 and stained for alkaline phosphatase (ALP) to demonstrate osteoblasts and for tartrate resistant acid phosphatase (TRAP) to visualize osteoclasts. Nonosteoclastic cells proliferated several-fold both on bioactive glass and on plastic, whereas osteoclasts and their precursors matured into multicellular giant cells and degenerated. Most cells on bioactive glass became ALP-positive, whereas on plastic the majority of cells remained ALP-negative. Osteoclasts survived on bioactive glass for 4-5 days, whereas on plastic they degenerated and disappeared after 3 days. Condensed nuclei indicating apoptosis were detected both in degenerating osteoclasts and osteoblasts. The surface of the bioactive glass reacted rapidly forming rounded pits, erosions, and cracks within 24 h in areas occupied by osteoblasts. Light microscopy and scanning electron micrographs demonstrated, however, a smooth surface below the cytoplasm of osteoclasts. This indicates that when applied on an intact bioactive glass surface, osteoclasts were unable to dissolve the glass material within this culture period.

In vitro comparison of clodronate, pamidronate and zoledronic acid effects on rat osteoclasts and human stem cell-derived osteoblasts.

In the present study we compared the first generation non-nitrogen-containing bisphosphonate, clodronate with second and third generation nitrogen-containing bisphosphonates, pamidronate and zoledronic acid in dynamic rat osteoclast resorption and apoptosis assays and in human mesenchymal stem cell-derived osteoblast assay. We found that due to high bisphosphonate-bone binding affinity, bone surface exposure to clodronate for 3 min. had maximal resorption inhibition. The mechanism of action of both clodronate and zoledronic acid involved osteoclast apoptosis, whereas pamidronate had only minor apoptotic effect at dosages, which readily inhibited resorption. Zoledronic acid was not metabolised into an intracellular ATP-analogue in vitro in contrast to clodronate. All bisphosphonates had a dose-dependent inhibitory effect on the human bone marrow mesenchymal stem cell (hMSC)-derived osteoblast calcium deposition. None of the compounds had inhibitory effect on hMSC differentiation. Zoledronic acid was the most potent of all three bisphosphonates in terms of both apoptosis induction and resorption inhibition. Zoledronic acid efficacy might thus use its capacity to trigger osteoclast apoptosis in an unknown, but similar manner to that of the non-nitrogen-containing bisphosphonates. It appears that zoledronic acid has properties of both bisphosphonate classes and could well be the first member of a new class of bisphosphonates, by definition.

Human mesenchymal stem cell derived osteoblasts degrade organic bone matrix in vitro by matrix metalloproteinases.

Some recent studies have suggested that cells of mesenchymal origin might participate in the organic bone matrix dissolution. In the present study, collagen synthesis and degradation by human mesenchymal stem cell (MSC) derived cells were studied at early stage of osteoblast differentiation using a special two-stage in vitro culture model. In this model, cells were cultured on bovine bone slices, which were first resorbed by osteoclasts. Synthesis of type I collagen was markedly enhanced when mesenchymal cells were cultured on bone matrix. After thorough osteoclast removal, MSC derived cells were capable of degrading the organic bone matrix, and caused a release of type I collagen degradation product (ICTP) into the culture medium. This was inhibited by matrix metalloproteinase (MMP) inhibitor, while cysteine proteinase inhibitor or estrogen had no inhibitory effect. Western blot analysis or gelatin zymography confirmed the presence of MMP-2, -8, -13 and -14, but not MMP-1 or -9, in the differentiated cells. 17beta-Estradiol was found to increase the expression of MMP-2 and -14 by these cells. Finally, scanning electron microscopy showed that the differentiating human MSCs were capable of degrading organic bone matrix remnants from the bottom of the resorption lacunae. These data support the hypothesis that collagen cleavage by the same cells that are subsequently responsible for bone formation is MMP mediated process and is an important step coupling bone formation into bone resorption.

Estrogen responsiveness of bone formation in vitro and altered bone phenotype in aged estrogen receptor-alpha-deficient male and female mice.

OBJECTIVE: Although the beneficial effects of estrogen on bone are well known, the roles of estrogen receptors (ERs) in mediating these effects are not fully understood. METHODS: To study the effects of long-term ER alpha deficiency, bone phenotype was studied in aged ER alpha knockout (ERKO) mice. In addition, ERKO osteoclasts and osteoblasts were cultured in vitro. DESIGN AND RESULTS: Histomorphometric analysis showed that the trabecular bone volume and thickness were significantly increased and the rate of bone formation enhanced in both male and female ERKO mice in comparison to the wild-type animals. In ERKO males, however, the bones were thinner and their maximal bending strengths decreased. Consistent with previous reports, the bones of knockout mice, especially of female mice, were shorter than those of wild-type mice. In addition, the growth plates were totally absent in the tibiae of aged ERKO females, whereas the growth plate cartilages were detectable in wild-type females as well as in all the males. Analysis of cultured bone marrow cells from 10- to 12-week-old mice demonstrated that 17 beta-estradiol could stimulate osteoblastic differentiation of bone marrow cells derived from ERKO mice relatively to the same extent as those derived from wild-type mice. This was demonstrated by increases in synthesis of type I collagen, activity of alkaline phosphatase and accumulation of calcium in cultures. Total protein content was, however, reduced in ERKO osteoblast cultures. CONCLUSIONS: These results show altered bone phenotype in ERKO mice and demonstrate the stimulatory effect of estrogen on osteoblasts even in the absence of full-length ER alpha.